Compositions and Methods of Vascular Injury Repair

ABSTRACT

The present invention relates to pharmaceutical compositions comprising a chemotactic hematopoietic stem cell product comprising an enriched population of CD34+ cells containing a subpopulation of cells having chemotactic activity, methods of preparing these compositions and use of these compositions to treat or repair vascular injury, including infarcted myocardium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/734,151, filed Nov. 7, 2005, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions comprising a chemotactichematopoietic stem cell product and methods of use thereof in repairinginjury caused by vascular insufficiency, including infarcted myocardium,as well as other vascular conditions similar to or related to vascularinsufficiency.

BACKGROUND OF THE INVENTION

Acute myocardial infarction remains common with a reported annualincidence of 1.1 million cases in the United States alone (Antman, E.M., Braunwald, E., Acute myocardial infarction, in Principles ofInternal Medicine, 15^(th) Ed., Braunwald, E. et al., Eds., New York:McGraw-Hill (2001)). Preclinical and clinical data demonstrate thatfollowing a myocardial infarction, the acute loss of myocardial musclecells and the accompanying peri-infarct zone hypo-perfusion result in acascade of events causing an immediate diminution of cardiac function,with the potential for long term persistence. The extent of myocardialcell loss is dependent on the duration of coronary artery occlusion,existing collateral coronary circulation and the condition of thecardiac microvasculature. Paul et al., Am. Heart J. 131: 710-15 (1996);Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990); Sheiban,I.e. al., J. Am. Coll. Cardiol. 38: 464-71 (2001); Braunwald E.,Bristow, M. R., Circulation 102: IV-14-23 (2000); Rich et al., Am. J.Med. 92:7-13 (1992); Ren et al., J. Histochem. Cytochem. 49: 71-79(2002); Hirai, T. et al., Circulation 79: 791-96 (1989); Ejiri, M. etal., J. Cardiology 20: 31-37 (1990). Because myocardial cells havevirtually no ability to regenerate, myocardial infarction leads topermanent cardiac dysfunction due to contractile-muscle cell loss andreplacement with nonfunctioning fibrotic scarring. Frangogiannis, N. G.,et al., Cardiovascular Res. 53(1): 31-47 (2002). Moreover, compensatoryhypertrophy of viable cardiac muscle leads to microvascularinsufficiency that results in further demise in cardiac function bycausing myocardial muscle hibernation and apoptosis of hypertrophiedmyocytes in the peri-infarct zone.

Among survivors of myocardial infarction, residual cardiac function isinfluenced most by the extent of ventricular remodeling (meaning changesin size, shape, and function, typically a decline in function, of theheart after injury). Alterations in ventricular topography (meaning theshape, configuration, or morphology of a ventricle) occur in bothinfarcted and healthy cardiac tissue after myocardial infarction.Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990).Ventricular dilatation (meaning a stretching, enlarging or spreading outof the ventricle) causes a decrease in global cardiac function and isaffected most by the infarct size, infarct healing and ventricular wallstresses. Recent efforts to minimize remodeling have been successful bylimiting infarct size through rapid reperfusion (meaning restoration ofblood flow) using thrombolytic agents and mechanical interventions,including, but not limited to, placement of a stent, along with reducingventricular wall stresses by judicious use of pre-load therapies andproper after-load management. Id. Regardless of these interventions, asubstantial percentage of patients experience clinically relevant andlong-term cardiac dysfunction after myocardial infarction. Sheiban, I.et al., J. Am. Coll. Cardiol. 38: 464-71 (2001). Despiterevascularization of the infarct related artery circulation andappropriate medical management to minimize ventricular wall stresses, asignificant percentage of patients experience ventricular remodeling,permanent cardiac dysfunction, and consequently remain at an increasedlifetime risk of experiencing adverse cardiac events, including death.Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer, M. A., Braunwald,E., Circulation 81: 1161-72 (1990).

At the cellular level, immediately following a myocardial infarction,transient generalized cardiac dysfunction uniformly occurs. In thesetting of a brief (i.e., lasting three minutes to five minutes)coronary artery occlusion, energy metabolism is impaired, leading todemonstrable cardiac muscle dysfunction that can persist for up to 48hours despite immediate reperfusion. This so-called “stunned myocardiumphenomenon” occurs subsequent to or after reperfusion and is thought tobe a result of reactive oxygen species. The process is transient and isnot associated with an inflammatory response. Frangogiannis, N. G., etal., Cardiovascular Res. 53(1): 31-47 (2002). After successfulrevascularization, significant recovery from stunning occurs withinthree to four days, although complete recovery may take much longer.Boli, R., Prog. Cardiovascular Disease 40(6): 477-515 (1998); Sakata, K.et al., Ann. Nucleic Med. 8: 153-57 (1994); Wollert, K. C. et al.,Lancet 364: 141-48 (2004).

Coronary artery occlusion of more significant duration, i.e., lastingmore than five minutes, leads to myocardial ischemia (i.e., aninsufficient blood flow to the heart's muscle mass) and is associatedwith a significant inflammatory response that begins immediately afterreperfusion and can last for up to several weeks. Frangogiannis, N. G.,et al., Cardiovascular Res. 53(1): 31-47 (2002); Frangogiannis, N. G. etal., Circulation 98: 687-798 (1998).

The inflammatory process following reperfusion is complex. Initially itcontributes to myocardial damage but later leads to healing and scarformation. This complex process appears to occur in two phases. In thefirst so-called “hot” phase (within the first five days), reactiveoxygen species (in the ischemic myocardial tissue) and complementactivation generate a signal chemotactic for leukocytes (chemotaxis isthe directed motion of a motile cell, organism or part towardsenvironmental conditions it deems attractive and/or away fromsurroundings it finds repellent) and initiate a cytokine cascade. Lefer,D. J., Granger, D. N., Am. J. Med. 4:315-23 (2000); Frangogiannis, N.G., et al., Circulation 7:699-710 (1998). Mast cell degranulation, tumornecrosis factor alpha (TNF-α) release, and increased interleukin-6(IL-6), intercellular adhesion molecule 1 (“ICAM-1” or CD-54, a receptortypically expressed on endothelial cells and cells of the immunesystem), selectin (L, E and P) and integrin (CD11a, CD11b and CD18)expression all appear to contribute to neutrophil accumulation anddegranulation in ischemic myocardium. Frangogiannis, N. G. et al.,Circulation 7: 699-710 (1998), Kurrelmeyer, K. M, et al., Proc. Nat'lAcad. Sci. 10: 5456-61 (2000); Lasky, L. A., Science 258: 964-69 (1992);Ma, X. L., et al., Circulation 88(2): 649-58 (1993); Simpson, P. J. etal., J. Clin. Invest. 2: 624-29 (1998). Neutrophils contributesignificantly to myocardial cell damage and death through microvascularobstruction and activation of neutrophil respiratory burst pathwaysafter ligand-specific adhesion to cardiac myocytes. Entman, M. L., etal., J. Clin. Invest. 4: 1335-45 (1992). During the “hot” phase,angiogenesis is inhibited due to the release of angiostatic substances,including interferon gamma-inducible protein (IP 10). Frangogiannis, N.G., et al., FASEB J. 15: 1428-30 (2001).

In the second phase, the cardiac repair process begins (about day 6 toabout day 14), which eventually leads to scar formation (about day 14 toabout day 21) and subsequent ventricular remodeling (about day 21 toabout day 90). Soon after reperfusion, monocytes infiltrate theinfarcted myocardium. Attracted by complement (C5a), transforming growthfactor B1 (“TGF-B1”) and monocyte chemotactic protein 1 (“MCP-1”),monocytes differentiate into macrophages that initiate the healingprocess by scavenging dead tissue, regulating extracellular matrixmetabolism, and inducing fibroblast proliferation. Birdshall, H. H., etal., Circulation 3: 684-92 (1997). Secretion of interleukin 10 (IL-10)by infiltrating lymphocytes also promotes healing by down-regulatinginflammatory cytokines and influencing tissue remodeling. Frangogiannis,N. G. et al., J. Immunol. 5:2798-2808 (2000). Mast cells also appear tobe involved in the later stages of myocardial repair by participating inthe formation of fibrotic scar tissue. Stem Cell Factor (SCF) is apotent attractor of mast cells. SCF mRNA has been shown to beup-regulated in ischemic myocardial segments in a canine model ofmyocardial infarction and thus may contribute to mast cell accumulationat ischemic myocardial sites. Frangogiannis, N. G. et al., Circulation98: 687-798 (1998). Mast cell products (including TGF-B, basicfibroblast growth factor (bFGF), vascular endothelial growth factor(VEGF) and gelatinases A and B) induce fibroblast proliferation,influence extracellular matrix metabolism, and induce angiogenesis.Fang, K. C., et al., J. Immunol. 162: 5528-35 (1999); Takeshi, S., etal., Cardiology 93: 168-74 (2000).

Following a myocardial infarction, neoangiogenesis occurs after the“hot” phase of the inflammatory process subsides (about day 5)coincident with rising levels of VEGF (VEGF peaks at about day 7 andgradually subsides to baseline at about day 14 to about day 21). Duringthis phase of the healing process, endothelial precursor cells (EPCs)are mobilized and recruited to the infarct site. Shinitani, S., et al.,Circulation 103: 2776-79 (2001). Without being limited by theory, it hasbeen suggested that the chemokine stromal cell derived factor-1 (SDF-1),which is the ligand for the CXCR-4 chemokine receptor expressed by CD34+cells, also plays a role in homing of cells to areas of ischemic damage.Ceredini, D. J., et al., Nature Medicine 10: 858-63 (2004); Askari, A.,et al., Lancet 362: 697-703 (2003); Yamaguchi, J. et al., Circulation107: 1322-34 (2003). While it is known that SDF-1 plays a role inhematopoiesis and is involved in migration, homing and survival ofhematopoietic progenitors, and while SDF-1 has been implicated inischemic neovascularization in vivo by augmenting EPC recruitment toischemic sites (Yamaguchi et al. Circulation 107:1322-1328 (2003),SDF-1's role in neoangiogenesis is not certain. There is suggestiveevidence implicating SDF-1. For example, SDF-1 gene expression isupregulated during hypoxia, a deficiency of oxygen in the tissues, byhypoxia inducible factor-1. Furthermore, CD34+ cells are capable ofhoming to areas of ischemia, rich in SDF-1, including infarctedmyocardium. Askari et al., Lancet 362: 697-703 (2003). Moreover,virtually all CD34⁺ CXCR-4⁺ cells co-express VEGF-2 and thereforemigrate in response to VEGF as well as SDF-1. Peichev M., et al., Blood95: 952-58 (2000). CD34⁺CXCR-4⁺ VEGF-1 cells, once recruited, arecapable of contributing to neoangiogenesis. Yamaguchi, J. et al.,Circulation 107: 1322-34 (2003).

To date, no ideal therapy exists for preventing the long term adverseconsequences of vascular insufficiency, particularly the significantvascular insufficiency that results in a myocardial infarction. Whilelarge vessel revascularization (meaning the successful placement of astent) seems promising, studies to date have shown such applications tobe insufficient in addressing increased demands posed by compensatorymyocardial hypertrophy. As a result, infarct extension and fibrousreplacement commonly occur, regardless of large vesselrevascularization, appropriate medical management of ventricular wallstresses, and potential natural, albeit suboptimal, CD34+ cell-mediatedneoangiogenesis (one of the theories relating to the underlying cause ofmyocardial infarction is that the ability to mobilize these cells may bebiologically limited).

Intense interest has developed in evaluating the ability of endothelialand myocardial precursor cells to limit damage to the myocardium afterinfarction and to limit or prevent ventricular remodeling. Significantpreclinical data and some clinical data demonstrate the safety andpotential of cell therapy using a variety of cell precursors(particularly hematopoietic cells) to contribute to neoangiogenesis,limited cardiac myogenesis (principally by fusion), and musclepreservation in the myocardial infarct zone. See, e.g., Jackson, et al.,J. Clin. Invest. 107: 1395-1402 (2001); Edelberg, J. M., et al., Cir.Res. 90: e89-e93 (2002); Schichinger, V. et al., New Engl. J. Med. 355(12): 1210-21 (2006) (using bone marrow-derived progenitor cells);Assmus, B. et al., New Engl. J. Med. 355 (12) 1222-32 (2006) (using bonemarrow-derived progenitor cells), but see Lunde, K. et al., New Eng. J.Med. 355 (12): 1199-209 (2006) (using bone marrow-derived progenitorcells).

Bone marrow consists of a variety of precursor and mature cell types,including hematopoietic cells (the precursors of mature blood cells) andstromal cells (the precursors of a broad spectrum of connective tissuecells), both of which appear to be capable of differentiating into othercell types. Wang, J. S. et al., J. Thorac. Cardiovasc. Surg. 122:699-705 (2001); Tomita, S. et al., Circulation 100 (Suppl. II): 247-256(1999); Saito, T. et al., Tissue Eng. 1: 327-43 (1995). Unmodified(i.e., not fractionated) marrow or blood-derived cells have been used inseveral clinical studies, for example, Hamano, K. et al., Japan Cir. J.65: 845-47 (2001); Strauer, B. E., et al., Circulation 106: 1913-18(2002); Assmus, et al., Circulation 106: 3009-3017 (2002); Dobert, N. etal., Eur. J. Nucl. Med. Mol. Imaging, 8: 1146-51 (2004); Wollert, K. C.et al., Lancet 364: 141-48 (2004). Since the mononuclear fraction ofbone marrow contains stromal cells, hematopoietic precursors, andendothelial precursors, the relative contribution of each of thesepopulations to the observed effects, if any, remains unknown.

CD34 is a hematopoietic stem cell antigen selectively expressed onhematopoietic stem and progenitor cells derived from human bone marrow,blood and fetal liver. Yin et al., Blood 90: 5002-5012 (1997); Miaglia,S. et al., Blood 90: 5013-21 (1997). Cells that express CD34 are termedCD34⁺. Stromal cells do not express CD34 and are therefore termed CD34⁻.CD34⁺ cells isolated from human blood may be capable of differentiatinginto cardiomyocytes, endothelial cells, and smooth muscle cells in vivo.See Yeh, et al., Circulation 108: 2070-73 (2003). CD34⁺ cells representapproximately 1% of bone marrow derived nucleated cells; CD34 antigenalso is expressed by immature endothelial cell precursors; matureendothelial cells do not express CD34+. Peichev, M. et al., Blood 95:952-58 (2000). In vitro, CD34+ cells derived from adult bone marrow giverise to a majority of the granulocyte/macrophage progenitor cells(CFU-GM), some colony-forming units-mixed (CFU-Mix) and a minorpopulation of primitive erythroid progenitor cells (burst forming units,erythrocytes or BFU-E). Yeh, et al., Circulation 108: 2070-73 (2003).CD34⁺ cells also may have the potential to differentiate into or tocontribute to the development of new myocardial muscle, albeit at lowfrequency.

Techniques have been developed using immunomagnetic bead separation toisolate a highly purified and viable population of CD34⁺ cells from bonenarrow mononuclear cells. See U.S. Pat. Nos. 5,536,475, 5,035,994,5,130,144, 4,965,205, the contents of each of which is incorporatedherein by reference. Two clinical studies support the clinicalapplication of bone marrow derived CD34+ cells after myocardialinfarction. See C. Stamm, et al., Lancet 361: 45-46 (2003); Herenstein,B. et al., Blood Supplement, Abs. 2696 (2004).

To date, however, no ideal therapy exists for preventing the long-termadverse consequences of vascular insufficiency, particularly vascularinsufficiency that produces myocardial infarction.

The present invention addresses the question of whether a compositioncomprising a chemotactic hematopoietic stem cell product comprising anenriched population of isolated CD34+ cells containing a subpopulationof cells having chemotactic activity can be manufactured, remain stablefor a commercially viable period, and be delivered to a subject in needthereof so that potent cells can home to and repair sites of vascularinsufficiency, including infarcted myocardium.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions comprisinga chemotactic hematopoietic stem cell product comprising an enrichedpopulation of CD34+ cells containing a subpopulation of cells havingchemotactic activity, methods of preparing these compositions and use ofthese compositions to treat or repair vascular injury, includinginfarcted myocardium. The present invention provides a pharmaceuticalcomposition for repairing a vascular injury in a subject comprising: (a)a therapeutically effective amount of a sterile isolated chemotactichematopoietic stem cell product, the chemotactic hematopoietic stem cellproduct comprising an enriched population of isolated CD34+ cells havinga subpopulation of potent cells having chemotactic activity; and (b) astabilizing amount of serum, wherein the composition is administered tothe subject parenterally through a catheter, and wherein thesubpopulation of potent cells having chemotactic activity when passedthrough the catheter remains potent. According to one embodiment, theenriched population of isolated CD34+ cells in the chemotactichematopoietic stem cell product of the composition is purified from bonemarrow acquired from the subject. According to another embodiment, theenriched population of isolated CD34+ cells is at least 70% pure.According to another embodiment, the enriched population of isolatedCD34+ cells is at least 70% viable for at least about 24 hours followingacquisition of the enriched population of isolated CD34+ cells.According to another embodiment, the enriched population of isolatedCD34+ cells is at least 70% viable for at least about 48 hours followingacquisition of the enriched population of isolated CD34+ cells.According to another embodiment, enriched population of isolated CD34+cells is at least 70% viable for at least about 72 hours followingacquisition of the enriched population of isolated CD34+ cells.According to another embodiment, the therapeutically effective amount ofthe chemotactic hematopoietic stem cell product comprises a minimumnumber of isolated CD34+ hematopoietic stem cells containing asubpopulation of at least 0.5×10⁶ potent CD34+ cells having CXCR-4mediated chemotactic activity. According to another embodiment, theenriched CD34+ cells are capable of forming hematopoietic colonies invitro for at least about 24 hours following acquisition of the enrichedCD34+ cells. According to another embodiment, the enriched CD34+ cellsare capable of forming hematopoietic colonies in vitro for at leastabout 48 hours following acquisition of the enriched CD34+ cells.According to another embodiment, the enriched CD34+ cells are capable offorming hematopoietic colonies in vitro for at least about 72 hoursfollowing acquisition of the enriched CD34+ cells. According to anotherembodiment, the serum is human serum autologous to the patient orsynthetic serum. According to another embodiment, the stabilizing amountof serum is at least about 10% (v/v). According to another embodiment,at least 2% of the chemotactic activity of the subpopulation of potentcells is retained by the subpopulation of potent cells for at least 24hours following acquisition of the enriched population of isolated CD34+cells. According to another embodiment, at least 2% of the chemotacticactivity of the subpopulation of potent cells is retained by thesubpopulation of potent cells for at least 48 hours followingacquisition of the enriched population of isolated CD34+ cells.According to another embodiment, at least 2% of the chemotactic activityof the subpopulation of potent cells is retained by the subpopulation ofpotent cells for at least 72 hours following acquisition of the enrichedpopulation of isolated CD34+ cells. According to another embodiment, theenriched population of isolated CD34+ cells containing a subpopulationof potent cells having chemotactic activity is at least 70% pure, is atleast about 70% viable, and is able to form hematopoietic colonies invitro for at least about 24 hours following acquisition of the enrichedpopulation. According to another embodiment, the enriched population ofisolated CD34+ cells containing a subpopulation of potent cells havingchemotactic activity is at least 70% pure, is at least about 70% viable,and is able to form hematopoietic colonies in vitro for at least about48 hours following acquisition of the enriched population. According toanother embodiment, the enriched population of isolated CD34+ cellscontaining a subpopulation of potent cells having chemotactic activityis at least 70% pure, is at least about 70% viable, and is able to formhematopoietic colonies in vitro for at least about 72 hours followingacquisition of the enriched population. According to another embodiment,the subject is a revascularized myocardial infarction patient. Accordingto another embodiment, the composition is administered through thecatheter intravascularly to an infarct-related artery. According toanother embodiment, the catheter is a flow control catheter. Accordingto another embodiment, the catheter is a balloon catheter. According toanother embodiment, the catheter has an internal diameter of at least0.36 mm. According to another embodiment, the composition isadministered through the catheter intravascularly by means of anintravascular delivery apparatus. According to another embodiment, thecomposition is administered through the catheter into myocardium.According to another embodiment, the pharmaceutical composition furthercomprises at least about 0.5% albumin. According to another embodiment,the pharmaceutical composition further includes at least one compatibleactive agent. According to another embodiment, the active agent isselected from the group consisting of an angiotensin converting enzymeinhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, ananti-anginal agent, a vasoactive agent, an anticoagulant agent, afibrinolytic agent, and a hypercholesterolemic agent. According toanother embodiment, sterility of the chemotactic hematopoietic cellproduct of the composition is confirmed by a method comprising thesteps: (a) centrifuging the cell product to form a separated cellproduct comprising a pellet comprising the enriched population ofisolated CD34+ cells and a supernatant; (b) removing the supernatant ofthe separated cell product without disturbing the cell pellet of theseparated cell product; (c) analyzing the sterility of the supernatantof the separated cell product; and (d) thereby determining the sterilityof the cell pellet of the separated cell product.

The present invention further provides a method of preparing apharmaceutical composition for intravascular delivery to a subjecthaving a vascular injury, the pharmaceutical composition comprising asterile chemotactic hematopoietic stem cell product comprising anenriched population of isolated CD34+ cells containing a subpopulationof potent cells having chemotactic activity, the method comprising thesteps: (a) acquiring a preparation comprising an enriched population ofpotent CD34+ cells from the subject under sterile conditions by achemotactic cell acquisition process, (b) optionally transporting thepreparation to a processing facility; (c) sterilely purifying CD34+cells containing a subpopulation of potent cells having chemotacticactivity from the preparation; (d) sterilely formulating the purifiedCD34+ cells containing a subpopulation of potent cells havingchemotactic activity to form the chemotactic hematopoietic cell product;(e) sterilely formulating the sterile chemotactic hematopoietic stemcell product to form a pharmaceutical composition; (e) assessingsterility of the pharmaceutical composition; (f) releasing the sterilepharmaceutical composition as eligible for infusion into the subject;(g) loading a therapeutic amount of the pharmaceutical composition intoan intravascular delivery apparatus; and (h) optionally transporting thedelivery apparatus containing the therapeutically effective amount ofthe sterile pharmaceutical composition to a cardiac catheterizationfacility for intravascular infusion into the subject. According to oneembodiment, the method optionally further comprises the step of storingthe acquired preparation between steps (a) and (b). According to anotherembodiment, purifying step (c) of the method is initiated within about12 hours to about 24 hours of completion of acquiring step (a).According to another embodiment, purifying step (c) of the method isinitiated within about 12 hours, about 13 hours, about 14 hours, about15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,or about 24 hours of completion of acquiring step (a). According toanother embodiment, releasing step (f) of the method proceeds only ifthe sterile formulated cell product is to be infused into the subjectwithin about 48 hours to about 72 hours of completion of acquiring step(a). According to another embodiment, releasing step (f) of the methodproceeds only if the sterile formulated cell product is to be infusedinto the subject within about 48 hours, about 49 hours, about 50 hours,about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours,about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours,about 69 hours, about 70 hours, about 71 hours or about 72 hours ofcompletion of acquiring step (a). According to another embodiment,purifying step (c) of the method is initiated within about 12 hours toabout 24 hours of completion of acquiring step (a), and releasing step(f) proceeds only if the sterile pharmaceutical composition is to beinfused into the subject within about 48 hours to about 72 hours ofcompletion of acquiring step (a). According to another embodiment,purifying step (c) of the method is initiated within about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, or about 24 hours of completion ofacquiring step (a), and releasing step (f) of the method proceeds onlyif the sterile pharmaceutical composition is to be infused into thesubject within about 48 hours, about 49 hours, about 50 hours, about 51hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours,about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours,about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69hours, about 70 hours, about 71 hours or about 72 hours of completion ofacquiring step (a). According to another embodiment, the chemotacticcell acquisition process in step (a) of the method is a mini-bone marrowharvesting technique. According to another embodiment, acquiring step(a) of the mini-bone marrow harvesting technique further comprises thesteps: (i) preloading harvesting syringes with heparin prior toharvesting bone marrow from the subject; (ii) aspirating the bone marrowfrom a left posterior iliac crest and a right posterior iliac crest ofthe subject using the harvesting syringes and a mini-bone marrow harvesttechnique to form harvested bone marrow; and (iii) infusing theharvested bone marrow into a collecting bag. According to anotherembodiment, the harvesting syringes in step (i) and the collecting bagin step (iii) contain a preservative free heparinized solution.According to another embodiment, the final concentration of heparin inthe preservative free heparinized solution is about 20 units per ml toabout 25 units per ml. According to another embodiment, transportingstep (b) of the method further comprises the steps: (i) placing theharvested bone marrow in a collection bag; (ii) placing the collectionbag in a secondary bag; (ii) preparing an open shipping container; (iii)placing the secondary bag containing the collection bag in the openshipping container; (iv) sealing the shipping container; and (v)shipping the shipping container to the processing facility. According toanother embodiment, the enriched population of isolated CD34+ cells instep (c) of the method is at least 70% pure. According to anotherembodiment, the enriched population of isolated CD34+ cells is at least70% viable for at least about 24 hours following acquisition of theenriched population of isolated CD34+ cells in step (a) of the method.According to another embodiment, the enriched population of isolatedCD34+ cells is at least 70% viable for at least about 48 hours followingacquisition of the enriched population of isolated CD34+ cells in step(a) of the method. According to another embodiment, the enrichedpopulation of isolated CD34+ cells is at least 70% viable for at leastabout 72 hours following acquisition of the enriched population ofisolated CD34+ cells in step (a) of the method. According to anotherembodiment, the enriched CD34+ cells are capable of forminghematopoietic colonies in vitro for at least about 24 hours followingacquisition of the enriched CD34+ cells in step (a) of the method.According to another embodiment, the enriched CD34+ cells are capable offorming hematopoietic colonies in vitro for at least about 48 hoursfollowing acquisition of the enriched CD34+ cells in step (a) of themethod. According to another embodiment, the enriched CD34+ cells arecapable of forming hematopoietic colonies in vitro for at least about 72hours following acquisition of the enriched CD34+ cells in step (a) ofthe method. According to another embodiment, at least 2% of thechemotactic activity of the subpopulation of potent cells is retained bythe subpopulation of potent cells for at least 24 hours followingacquisition of the enriched population of isolated CD34+ cells in step(a) of the method. According to another embodiment, at least 2% of thechemotactic activity of the subpopulation of potent cells is retained bythe subpopulation of potent cells for at least 48 hours followingacquisition of the enriched population of isolated CD34+ cells in step(a) of the method. According to another embodiment, at least 2% of thechemotactic activity of the subpopulation of potent cells is retained bythe subpopulation of potent cells for at least 72 hours followingacquisition of the enriched population of isolated CD34+ cells in step(a) of the method. According to another embodiment, the enrichedpopulation of isolated CD34+ cells containing a subpopulation of potentcells having chemotactic activity is at least 70% pure, is at leastabout 70% viable, and is able to form hematopoietic colonies in vitrofor at least about 24 hours following acquisition of the enrichedpopulation of isolated CD34+ cells in step (a) of the method. Accordingto another embodiment, the enriched population of isolated CD34+ cellscontaining a subpopulation of potent cells having chemotactic activityis at least 70% pure, is at least about 70% viable, and is able to formhematopoietic colonies in vitro for at least about 48 hours followingacquisition of the enriched population of isolated CD34+ cells in step(a) of the method. According to another embodiment, the enrichedpopulation of isolated CD34+ cells containing a subpopulation of potentcells having chemotactic activity is at least 70% pure, is at leastabout 70% viable, and is able to form hematopoietic colonies in vitrofor at least about 72 hours following acquisition of the enrichedpopulation of isolated CD34+ cells in step (a) of the method. Accordingto another embodiment, the therapeutically effective amount of thechemotactic hematopoietic stem cell product in the pharmaceuticalcomposition of step (g) of the method comprises a minimum number ofCD34+ hematopoietic stem cells containing a subpopulation of at least0.5×10⁶ CD34+ potent cells having CXCR-4 mediated chemotactic activity.According to another embodiment, the confirming sterility step (e) ofthe method further comprises the steps of (i) centrifuging theformulated cell product to form separated cell product comprising a cellpellet comprising the enriched population of isolated CD34+ cells and asupernatant, (ii) sterilely removing the supernatant of the separatedcell product without disturbing the cell pellet of the separated cellproduct; (iii) analyzing whether the supernatant of the separated cellproduct is contaminated by a microbe; and (iv) thereby determining thesterility of the cell pellet of the separated cell product. According toanother embodiment, the intravascular delivery apparatus of step (g) ofthe method comprises (i) an infusion syringe attached to a sterilefour-way stopcock, wherein the infusion syringe contains thepharmaceutical composition; (ii) a flushing syringe attached to thesterile four-way stopcock containing a flushing solution; and (iii) acatheter attached to the delivery apparatus by the sterile four-waystopcock for delivery of the pharmaceutical composition to the subject.According to another embodiment, the catheter is a flow controlcatheter. According to another embodiment, the catheter is a balloondilatation catheter. According to another embodiment, the catheter hasan internal diameter of at least 0.36 mm. According to anotherembodiment, the subject in need thereof is a revascularized myocardialinfarction patient. According to another embodiment, the pharmaceuticalcomposition is delivered to the subject by infusion of the compositionthrough the catheter to an infarct related artery. According to anotherembodiment, the pharmaceutical composition is administered through thecatheter into myocardium.

Moreover, the present invention provides a method of treating orrepairing a vascular injury in a subject in need thereof, the methodcomprising the step of administering to the subject parenterally througha catheter a sterile pharmaceutical composition comprising: (a) atherapeutically effective amount of a sterile chemotactic hematopoieticstem cell product, the chemotactic hematopoietic stem cell productcomprising an enriched population of isolated CD34+ cells containing asubpopulation of potent cells having chemotactic activity; and (b) astabilizing amount of serum, wherein the subpopulation of potent cellshaving chemotactic activity when passed through the catheter remainspotent. According to one embodiment, the enriched population of isolatedCD34+ cells is at least 70% pure. According to another embodiment, theenriched population of isolated CD34+ cells is at least 70% viable forat least about 24 hours following acquisition of the enriched populationof isolated CD34+ cells. According to another embodiment, the enrichedpopulation of isolated CD34+ cells is at least 70% viable for at leastabout 48 hours following acquisition of the enriched population ofisolated CD34+ cells. According to another embodiment, the enrichedpopulation of isolated CD34+ cells is at least 70% viable for at leastabout 72 hours following acquisition of the enriched population ofisolated CD34+ cells. According to another embodiment, thetherapeutically effective amount of the chemotactic hematopoietic stemcell product comprises a minimum number of isolated CD34+ hematopoieticstem cells containing a subpopulation of at least 0.5×10⁶ potent CD34+cells having CXCR-4 mediated chemotactic activity. According to anotherembodiment, the enriched CD34+ cells are capable of forminghematopoietic colonies in vitro for at least about 24 hours followingacquisition of the enriched CD34+ cells. According to anotherembodiment, the enriched CD34+ cells are capable of forminghematopoietic colonies in vitro for at least about 48 hours followingacquisition of the enriched CD34+ cells. According to anotherembodiment, the enriched CD34+ cells are capable of forminghematopoietic colonies in vitro for at least about 72 hours followingacquisition of the enriched CD34+ cells. According to anotherembodiment, at least 2% of the chemotactic activity of the subpopulationof potent cells is retained by the subpopulation of potent cells for atleast 24 hours following acquisition of the enriched population ofisolated CD34+ cells. According to another embodiment, at least 2% ofthe chemotactic activity of the subpopulation of potent cells isretained by the subpopulation of potent cells for at least 48 hoursfollowing acquisition of the enriched population of isolated CD34+cells. According to another embodiment, at least 2% of the chemotacticactivity of the subpopulation of potent cells is retained by thesubpopulation of potent cells for at least 72 hours followingacquisition of the enriched population of isolated CD34+ cells.According to another embodiment, the enriched population of isolatedCD34+ cells containing a subpopulation of potent cells havingchemotactic activity is at least 70% pure, is at least about 70% viable,and is able to form hematopoietic colonies in vitro for at least about24 hours following acquisition of the enriched population. According toanother embodiment, the enriched population of isolated CD34+ cellscontaining a subpopulation of potent cells having chemotactic activityis at least 70% pure, is at least about 70% viable, and is able to formhematopoietic colonies in vitro for at least about 48 hours followingacquisition of the enriched population. According to another embodiment,the enriched population of isolated CD34+ cells containing asubpopulation of potent cells having chemotactic activity is at least70% pure, is at least about 70% viable, and is able to formhematopoietic colonies in vitro for at least about 72 hours followingacquisition of the enriched population. According to another embodiment,the serum is human serum autologous to the subject or a synthetic serum.According to another embodiment, the stabilizing amount of serum is atleast about 10% (v/v). According to another embodiment, the subject is arevascularized myocardial infarction patient. According to anotherembodiment, the method further comprising the step of delivering thecomposition intravascularly to an infarct related artery by means of anintravascular delivery apparatus. According to another embodiment, theintravascular delivery apparatus comprises (i) an infusion syringeattached to a sterile four-way stopcock, wherein the infusion syringecontains the pharmaceutical composition; (ii) a flushing syringeattached to the sterile four-way stopcock containing a flushingsolution; and (iii) a catheter attached to the delivery apparatus by thesterile four-way stopcock for delivery of the pharmaceutical compositionto the subject. According to another embodiment, the catheter is a flowcontrol catheter. According to another embodiment, the catheter is aballoon dilatation catheter. According to another embodiment, thecatheter has an internal diameter of at least 0.36 mm. According toanother embodiment, the pharmaceutical composition is delivered to thesubject during a specific time interval defined by a first time and asecond time. According to another embodiment, the first time is at leastabout 5 days post-infarction and the second time is less than about 14days post-infarction. According to another embodiment, the first time isafter peak inflammatory cytokine cascade production in the infractedarea. According to another embodiment, the second time is beforemyocardial scar formation in the infracted area. According to anotherembodiment, the composition is administered through the catheterintravascularly by means of an intravascular delivery apparatus.According to another embodiment, the composition is administered throughthe catheter into myocardium. According to another embodiment, thepharmaceutical composition further includes at least one compatibleactive agent. According to another embodiment, the active agent isselected from the group consisting of an angiotensin converting enzymeinhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, ananti-anginal agent, a vasoactive agent, an anticoagulant agent, afibrinolytic agent, and a hypercholesterolemic agent.

In addition, the present invention provides a method of assessingsterility of a clinically useful isolated cell product having a limitedvolume, the method comprising the steps: (a) sterilely separatingcellular components of the isolated cell product from noncellularcomponents; and (b) testing the noncellular components for microbialcontamination, thereby determining the sterility of the clinicallyuseful isolated cell product. According to one embodiment, in step (a)of the method the cellular components are separated from noncellularcomponents by centrifugation. According to another embodiment, thecellular components form a cell pellet upon centrifugation of theisolated cell product. According to another embodiment, the noncellularcomponents form a supernatant upon centrifugation of the isolated cellproduct. According to another embodiment, the isolated cell product is achemotactic hematopoietic stem cell product. According to anotherembodiment, the chemotactic hematopoietic stem cell product comprises anenriched population of isolated CD34+ cells containing a subpopulationof potent cells having CXCR-4 mediated chemotactic activity. Accordingto another embodiment, after sterility of the supernatant is confirmed,a therapeutically effective amount of the sterile chemotactichematopoietic stem cell product is administered to a subjectintravascularly to treat or repair a vascular injury. According toanother embodiment, the sterile chemotactic hematopoietic stem cellproduct is administered through a catheter. According to anotherembodiment, the subject is a revascularized myocardial infarctionpatient. According to another embodiment, the vascular injury is avascular insufficiency. According to another embodiment, the vascularinsufficiency affects at least one coronary artery.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that the functional viability of the chemotactichematopoietic cell product of the present invention at 72 hours isequivalent to that at 48 hours.

FIG. 2 shows the migratory efficiency of the formulated chemotactichematopoietic stem cell product comprising CD34+ cells of the presentinvention.

FIG. 3 shows the improved stability of CD34+ cells formulated in humanserum.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions set forth the parameters of the presentinvention.

As used herein, the term “angiogenesis” refers to the process offormation and development of blood vessels.

The term “c-kit” refers to a protein on the surface of some cells thatbinds to stem cell factor (a substance that causes certain types ofcells to grow). Altered forms of this receptor may be associated withsome types of cancer.

The term “cardiac biomarkers” refers to enzymes, proteins and hormonesassociated with heart function, damage or failure that are used fordiagnostic and prognostic purposes. Different biomarkers have differenttimes that their levels rise, peak, and fall within the body, allowingthem to be used not only to track the progress of a heart attack but toestimate when it began and to monitor for recurrence. Some of the testsare specific for the heart while others are also elevated with skeletalmuscle damage Current cardiac biomarkers include, but are not limitedto, CK (creatine phosphokinase or creatine kinase) and CK-MB (creatinekinase-myoglobin levels (to help distinguish between skeletal and heartmuscle)), troponin (blood levels of troponin I or T will remain high for1-2 weeks after a heart attack; troponin is not generally affected bydamage to other muscles), myoglobin (to determine whether muscle,particularly heart muscle, has been injured), and BNP (brain natriureticpeptide) or NT-proBNP (N-terminal prohormone brain natriuretic peptide(to help diagnose heart failure and grade the severity of that heartfailure).

The term “cardiac catheterization” refers to a procedure in which acatheter is passed through an artery to the heart, and into a coronaryartery. This procedure produces angiograms (i.e., x-ray images) of thecoronary arteries and the left ventricle, the heart's main pumpingchamber, can be used to measure pressures in the pulmonary artery, andto monitor heart function.

The term “CD34⁺ cells” as used herein refers to hematopoietic stem andprogenitor cells derived from human bone marrow that “are positive for”i.e., “express”, a hematopoietic stem cell antigen, at least asubpopulation of which express CXCR4, and that can migrate to areas ofinjury.

The term “CD38” refers to a protein marker present on macrophages,dendritic cells, and activated B and NK cells, which may mediate theadhesion between lymphocytes and endothelial cells.

The terms “CD45” and “common leukocyte antigen” refer to a proteintyrosine phosphatase (PTP) located in hematopoietic cells excepterythrocytes and platelets.

The term “CD59” refers to a glycosylphosphatidylinositol (GPI)-linkedmembrane glycoprotein which protects human cells fromcomplement-mediated lysis.

The term “chemotaxis” refers to the directed motion of a motile cell orpart towards environmental conditions it deems attractive and/or awayfrom surroundings it finds repellent.

The term “CXCR-4” as used herein refers to a G-protein-linked chemokinereceptor.

The term “cytokine” as used herein refers to small soluble proteinsubstances secreted by cells which have a variety of effects on othercells. Cytokines mediate many important physiological functionsincluding growth, development, wound healing, and the immune response.They act by binding to their cell-specific receptors located in the cellmembrane, which allows a distinct signal transduction cascade to startin the cell, which eventually will lead to biochemical and phenotypicchanges in target cells. Generally, cytokines act locally. They includetype I cytokines, which encompass many of the interleukins, as well asseveral hematopoietic growth factors; type II cytokines, including theinterferons and interleukin-10; tumor necrosis factor (“TNF”)-relatedmolecules, including TNF and lymphotoxin; immunoglobulin super-familymembers, including interleukin 1 (“IL-1”); and the chemokines, a familyof molecules that play a critical role in a wide variety of immune andinflammatory functions. The same cytokine can have different effects ona cell depending on the state of the cell. Cytokines often regulate theexpression of, and trigger cascades of, other cytokines.

The term “colony stimulating factor” refers to a cytokine responsiblefor controlling the production of white blood cells. Types includegranulocyte colony stimulating factor (G-CSF), macrophage colonystimulating factor (M-CSF), and granulocyte macrophage colonystimulating factor (GM-CSF).

The term “hematopoietic stem cell” refers to a cell isolated from theblood or from the bone marrow that can renew itself, differentiate to avariety of specialized cells, mobilize out of the bone marrow into thecirculating blood, and can undergo programmed cell death (apoptosis). Insome embodiments of the present invention, hematopoietic stem cellsderived from human subjects express at least one type of cell surfacemarker, including, but not limited to, CD34, CD38, HLA-DR, c-kit, CD59,Sca-1, Thy-1, and/or CXCR-4, or a combination thereof.

“HLA-DR” refers to a human class II histocompatibility antigen presenton several cell types, including antigen-presenting cells, B cells,monocytes, macrophages, and activated T cells.

The term “interleukin” as used herein refers to a cytokine secreted bywhite blood cells as a means of communication with other white bloodcells.

The terms “VEGF-1” or “vascular endothelial growth factor-1” are usedinterchangeably to refer to a cytokine that mediates numerous functionsof endothelial cells including proliferation, migration, invasion,survival, and permeability. VEGF is critical for angiogenesis.

The term “chemokine” as used herein refers to a class of chemotacticcytokines that signal leukocytes to move in a specific direction. Theterm “chemotactic” refers to movement or orientation of a cell along achemical concentration gradient either toward or away from a chemicalstimulus.

The term “complete blood count” (CBC) refers to a laboratory test thatprovides detailed information about the amount and the quality of eachof the blood cells types. It usually includes a measurement of each ofthe three major blood cells (red blood cells, white blood cells, andplatelets) and a measure of the hemoglobin and hematocrit. “Hemoglobin”(HGB) refers to the number of grams of hemoglobin in a deciliter ofblood (g/dL). Normal hemoglobin levels in healthy adult human subjectsare about 14 g/dL to about 18 g/dL for men and about 12 g/dL to about 16g/dL for women. As a rough guideline, hemoglobin generally should beabout one-third the hematocrit. “Red Blood Cell Count” (RBC) refers tothe total number of red blood cells in a quantity of blood. Normalranges in human subjects are about 4.5 million cells/mm³ to about 6.0million cells/mm³ for men and about 4.0 million cells/mm³ to about 5.5million cells/mm³ for women. “White Blood Cell Count” (WBC) refers tothe total number of white blood cells or leukocytes in a quantity ofblood. Normal ranges in human subjects are about 4.3×103 cells/mm³ toabout 10.8×103 cells/mm³. “Hematocrit” (HCT) refers to the proportion ofred blood cells as a percentage of total blood volume. A normalhematocrit for human subjects is about 40% to about 55% for men andabout 35% to about 45% for women.

The term “disease” or “disorder”, as used herein, refers to animpairment of health or a condition of abnormal functioning. The term“syndrome,” as used herein, refers to a pattern of symptoms indicativeof some disease or condition. The term “condition”, as used herein,refers to a variety of health states and is meant to include disordersor diseases caused by any underlying mechanism or disorder, injury, andthe promotion of healthy tissues and organs.

As used herein, the term “inflammation” refers to a response toinfection and injury in which cells involved in detoxification andrepair are mobilized to the compromised site by inflammatory mediators.Inflammation is often characterized by a strong infiltration ofleukocytes at the site of inflammation, particularly neutrophils(polymorphonuclear cells). These cells promote tissue damage byreleasing toxic substances at the vascular wall or in uninjured tissue.

Regardless of the initiating agent, the physiologic changes accompanyingacute inflammation encompass four main features: (1) vasodilation, whichresults in a net increase in blood flow, is one of the earliest sphysical responses to acute tissue injury; (2) in response toinflammatory stimuli, endothelial cells lining the venules contract,widening the intracellular junctions to produce gaps, leading toincreased vascular permeability which permits leakage of plasma proteinsand blood cells out of blood vessels; (3) inflammation often ischaracterized by a strong infiltration of leukocytes at the site ofinflammation, particularly neutrophils (polymorphonuclear cells). Thesecells promote tissue damage by releasing toxic substances at thevascular wall or in uninjured tissue; and (4) fever, produced bypyrogens released from leukocytes in response to specific stimuli.

During the inflammatory process, soluble inflammatory mediators of theinflammatory response work together with cellular components in asystemic fashion in the attempt to contain and eliminate the agentscausing physical distress. The terms “inflammatory” orimmuno-inflammatory” as used herein with respect to mediators refers tothe molecular mediators of the inflammatory process. These soluble,diffusible molecules act both locally at the site of tissue damage andinfection and at more distant sites. Some inflammatory mediators areactivated by the inflammatory process, while others are synthesizedand/or released from cellular sources in response to acute inflammationor by other soluble inflammatory mediators. Examples of inflammatorymediators of the inflammatory response include, but are not limited to,plasma proteases, complement, kinins, clotting and fibrinolyticproteins, lipid mediators, prostaglandins, leukotrienes,platelet-activating factor (PAF), peptides and amines, including, butnot limited to, histamine, serotonin, and neuropeptides, proinflammatorycytokines, including, but not limited to, interleukin-1, interleukin-4,interleukin-6, interleukin-8, tumor necrosis factor (TNF),interferon-gamma, and interleukin 12.

The term “in-date” refers to the time interval between completion ofacquiring from the subject a preparation comprising an enrichedpopulation of potent CD34+ cells from a subject under sterile conditionsand initiating sterilely purifying potent CD34+ cells from thepreparation. The term “out-date” refers to the time interval betweencompletion of acquiring from the subject a preparation comprising anenriched population of potent CD34+ cells from a subject under sterileconditions and infusing the formulated pharmaceutical compositioncomprising a chemotactic hematopoietic cell product into the subject.

The terms “infuse” or “infusion” as used herein refer to theintroduction of a fluid other than blood into a blood vessel of asubject, including humans, for therapeutic purposes.

The “infusion solution” of the present invention without autologousserum contains phosphate buffered saline (PBS) supplemented with 25 USPunits/ml of heparin and 1% human serum albumin (HSA). In someembodiments, the infusion solution is supplemented with serum. In someembodiments, the serum is autologous.

The term “injury” refers to damage or harm caused to the structure orfunction of the body of a subject caused by an agent or force, which maybe physical or chemical. The term “vascular injury” refers to injury tothe vasculature (i.e., the vascular network, meaning the network ofblood vessels or ducts that convey fluids, such as, without limitation,blood or lymph).

The term “macrophage” as used herein refers to a mononuclear, activelyphagocytic cell arising from monocytic stem cells in the bone marrow.These cells are widely distributed in the body and vary in morphologyand motility. Phagocytic activity is typically mediated by serumrecognition factors, including certain immunoglobulins and components ofthe complement system, but also may be nonspecific. Macrophages also areinvolved in both the production of antibodies and in cell-mediatedimmune responses, particularly in presenting antigens to lymphocytes.They secrete a variety of immunoregulatory molecules.

The term “microbe” or “microorganism” are used interchangeably herein torefer to an organism too small to be seen clearly with the naked eye,including, but not limited to, microscopic bacteria, fungi (molds),algae, protozoa, and viruses.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The term “myocardial infarction” refers to death or permanent damage toheart muscle. Most heart attacks arc caused by blockage of coronaryarteries that interrupts flow of blood and oxygen to the heart muscle,leading to death of heart cells in that area. The damaged heart muscleloses its ability to contract, leaving the remaining heart muscle tocompensate for the weakened area. The present invention includes stepsrelated to evaluating the suitability of subjects for treatmentaccording to the present invention by using tests to look at the size,shape, and function of the heart as it is beating, to detect changes tothe rhythm of the heart, and to detect and evaluate damaged tissues andblocked arteries. Examples of such tests include, but are not limitedto, electrocardiography, echocardiography, coronary angiography, andnuclear ventriculography. Cardiac biomarkers also are used to evaluatethe suitability of subjects for treatment according to the presentinvention.

As used herein, the term “potent” or “potency” refers to the necessarybiological activity of the chemotactic hematopoietic stem cell productof the present invention, i.e., potent cells of the present inventionremain viable, are capable of mediated mobility, and are able to grow,i.e., to form hematopoietic colonies in an in vitro CFU assay.

The term “progenitor cell” as used herein refers to an immature cell inthe bone marrow that can be isolated by growing suspensions of marrowcells in culture dishes with added growth factors. Progenitor cellsmature into precursor cells that mature into blood cells. Progenitorcells are referred to as colony-forming units (CFU) or colony-formingcells (CFC). The specific lineage of a progenitor cell is indicated by asuffix, such as, but not limited to, CFU-E (erythrocytic), CFU-GM(granulocytic/macrophage), and CFU-GEMM (pluripotent hematopoieticprogenitor).

The term “repair” as used herein as a noun refers to any correction,reinforcement, reconditioning, remedy, making up for, making sound,renewal, mending, patching, or the like that restores function. Whenused as a verb, it means to correct, to reinforce, to recondition, toremedy, to make up for, to make sound, to renew, to mend, to patch or tootherwise restore function. In some embodiments “repair” includes fullrepair and partial repair.

The term “Sca-1” or “stem cell antigen-1” refers to a surface proteincomponent in a signaling pathway that affects the self-renewal abilityof mesenchymal stem cells.

The term “stem cells” refers to undifferentiated cells having highproliferative potential with the ability to self-renew that can generatedaughter cells that can undergo terminal differentiation into more thanone distinct cell phenotype.

The term “stent” is used to refer to small tube used to prop open anartery. The stent is collapsed to a small diameter, put over a ballooncatheter, inserted through a main artery in the groin (femoral artery)or arm (brachial artery) and threaded up to the narrowed/blocked sectionof the artery. When it reaches the right location, the balloon isinflated slightly to push any plaque out of the way and to expand theartery (balloon angioplasty). When the balloon is inflated, the stentexpands, locks in place and forms a scaffold to hold the artery open.The stent stays in the artery permanently. In certain subjects, a stentreduces the renarrowing that occurs after balloon angioplasty or otherprocedures that use catheters. A stent also may help restore normalblood flow and keep an artery open if it has been torn or injured by theballoon catheter. Reclosure (restenosis) is a problem with the stentprocedure. Drug-eluting stents are stents coated with drugs that areslowly released. These drugs may help keep the blood vessel fromreclosing.

The term “subject” as used herein includes animal species of mammalianorigin, including humans.

The term “Thy-1” refers to the Ig superfamily cell surface glycoproteinThy-1 expressed on immune cells and neurons of rodents and humans, whichis hypothesized to function in cell adhesion and signal transduction inT cell differentiation, proliferation, and apoptosis.

As used herein the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition, substantially preventing the appearance of clinical oraesthetical symptoms of a condition, and protecting from harmful orannoying stimuli.

Compositions of the Present Invention

In one aspect of the present invention, the hematopoietic stem cells ofthe present invention can migrate, meaning that they can move from oneplace, location or area to another. In one embodiment, hematopoieticstem cell migration is driven by chemotaxis.

The present invention provides pharmaceutical compositions and methodsfor repair of injury caused by vascular insufficiency. The terms“formulation” and “composition” are used interchangeably herein to referto a product of the present invention that comprises all active andinert ingredients. The term “active” refers to the ingredient, componentor constituent of the compositions of the present invention responsiblefor the intended therapeutic effect. The terms “pharmaceuticalformulation” or “pharmaceutical composition” as used herein refer to aformulation or composition that is employed to prevent, reduce inintensity, cure or otherwise treat a target condition or disease.

The pharmaceutical composition for repair of vascular injury of thepresent invention comprises a chemotactic hematopoietic stem cellproduct comprising an enriched population of CD34+ cells containing asubpopulation of cells having chemotactic activity. In some embodiments,this chemotactic activity is mediated by SDF-1, VEGF, and/or CXCR-4.According to one embodiment, the chemotactic hematopoeitic stem cellproduct is prepared by isolating or purifying CD34+ hematopoietic stemcells from bone marrow harvested from the subject. According to thepresent invention, the chemotactic hematopoietic stem cell productenriched for CD34+ cells contains at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90% or atleast about 95% pure CD34+ cells. In another embodiment, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90% or at least about 95% of the CD34+ cells are viable forat least about 24, at least about 48 hours, or at least about 72 hoursfollowing acquisition of the enriched population of CD34+ cells. Inanother embodiment, the CD34+ cells can form hematopoietic colonies invitro for at least about 24, at least about 48 hours, or at least about72 hours following acquisition of the enriched population of CD34+cells.

CD34+ cells can be enriched/selected by any techniques known to theskilled artisan. For example, in some embodiments, the population ofbone marrow cells comprising CD34+ cells is enriched for cellsexpressing CD34 cell antigen and CXCR4 cell antigen by fluorescenceactivated cell sorting (FACS). In some embodiments, CD34+ cells in thebone marrow are enriched/selected by positive or negativeimmunoseparation techniques. In some embodiments, isolation and/orpurification of hematopoietic stem cells from the bone marrow is basedon cell fractionation methods based on size and cell density, efflux ofmetabolic dyes, or resistance to cytotoxic agents. In one embodiment,for example, CD34+ cells in the bone marrow are enriched/selected usinga monoclonal anti-CD34 antibody and an immunomagnetic separationtechnique.

The selected CD34+ cells can be identified, quantified and characterizedby techniques known in the art. For example, in some embodiments, thepercentage of CD34+ cells in the bone marrow and in the chemotactichematopoietic stem cell product can be determined by FACS analysis. Inanother embodiment, CD34 protein expression is quantified by Westernblot. The term “Western blot” refers to a method for identifyingproteins in a complex mixture; proteins are separatedelectrophoretically in a gel medium; transferred from the gel to aprotein binding sheet or membrane; and the sheet or membrane containingthe separated proteins exposed to specific antibodies which bind to,locate, and enable visualization of protein(s) of interest. For example,monoclonal anti-CD34 antibody can be used to detect CD34 protein adheredto a membrane in situ.

In another embodiment, the expression of CD34 mRNA and DNA in theisolated CD34+ cells can be quantified. The term “Northern blot” as usedherein refers to a technique in which RNA from a specimen is separatedinto its component parts on a gel by electrophoresis and transferred toa specifically modified paper support so that the mRNA is fixed in itselectrophoretic positions. CD34 related sequences are identified usingprobes comprising a reporter molecule, such as, without limitation, aradioactive label. In another embodiment, the level of CD34 and/or CXCR4expression is/are determined by quantitative or semi-quantitative PCR orreverse transcriptase PCR (“RT-PCR”) techniques. The abbreviation “PCR”refers to polymerase chain reaction, which is a technique for amplifyingthe quantity of DNA, thus making the DNA easier to isolate, clone andsequence. See, e.g., U.S. Pat. Nos. 5,656,493, 5,333,675, 5,234,824, and5,187,083, each of which is incorporated herein by reference.

In another embodiment, the selected CD34+ hematopoietic stem cells ofthe chemotactic hematopoietic stem cell product of the present inventioncontain a subpopulation of CD34+ cells having CXCR-4 mediatedchemotactic activity. In a preferred embodiment, the hematopoietic stemcell product of the present invention comprises a minimum number ofisolated CD34+ hematopoietic stem cells such that a subpopulation of atleast 0.5×10⁶ CD34+ cells having CXCR-4 mediated chemotactic activity ispresent. In another embodiment, at least about 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, or 34% of theCXCR-4 mediated chemotactic activity of the CD34+ cells is retained forat least 24 hours, at least 48 hours, or at least 72 hours followingacquisition of the enriched population of CD34+ cells. In anotherembodiment, at least an average of about 17% of the CXCR-4 mediatedchemotactic activity of the CD34+ cells is retained for at least 24hours, at least 48 hours, or at least 72 hours following acquisition ofthe enriched population of CD34+ cells. In another embodiment, the CD34+cells in the chemotactic hematopoietic cell product retain at leastabout 2% of the CXCR-4 mediated chemotactic activity for at least 72hours following acquisition of the enriched population of CD34+ cells.

The pharmaceutical composition of the present invention furthercomprises serum at a concentration of at least 10% by volume of thecomposition. In one embodiment, the serum is autologous. In anotherembodiment, the serum is a synthetic or recombinant serum. The minimumconcentration of serum present in the composition is at least about 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% expressedas ml/100 cc final volume of the composition. The maximum concentrationof serum present in the composition of the present invention is about70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%expressed as ml/100 cc final volume of the composition.

In some embodiments, the composition of the present invention may beformulated with an excipient, carrier or vehicle including, but notlimited to, a solvent. The terms “excipient”, “carrier”, or “vehicle” asused herein refers to carrier materials suitable for formulation andadministration of the Chemotactic hematopoietic stem cell productdescribed herein. Carriers and vehicles useful herein include any suchmaterials known in the art which are nontoxic and do not interact withother components. As used herein the phrase “pharmaceutically acceptablecarrier” refers to any substantially non-toxic carrier useable forformulation and administration of the composition of the presentinvention in which the chemotactic hematopoietic stem cell product ofthe present invention will remain stable and bioavailable.

The pharmaceutically acceptable carrier must be of sufficiently highpurity and of sufficiently low toxicity to render it suitable foradministration to the mammal being treated. It further should maintainthe stability and bioavailability of an active agent. Thepharmaceutically acceptable carrier can be liquid or solid and isselected, with the planned manner of administration in mind, to providefor the desired bulk, consistency, etc., when combined with an activeagent and other components of a given composition. For example, thepharmaceutically acceptable carrier can be, without limitation, abinding agent (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose, etc.), a filler (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate,etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.), a disintegrant (e.g., starch, sodium starchglycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate,etc.). Other suitable pharmaceutically acceptable carriers for thecompositions of the present invention include, but are not limited to,water, salt solutions, alcohols, polyethylene glycols, gelatins,amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins,hydroxymethylcelluloses, polyvinylpyrrolidones and the like. Suchcarrier solutions also can contain buffers, diluents and other suitableadditives. The term “buffer” as used herein refers to a solution orliquid whose chemical makeup neutralizes acids or bases without asignificant change in pH. Examples of buffers envisioned by the presentinvention include, but are not limited to, Dulbecco's phosphate bufferedsaline (PBS), Ringer's solution, 5% dextrose in water (D5W),normal/physiologic saline (0.9% NaCl). In some embodiments, the infusionsolution is isotonic to subject tissues. In some embodiments, theinfusion solution is hypertonic to subject tissues. Compositions of thepresent invention that are for parenteral administration can includepharmaceutically acceptable carriers such as sterile aqueous solutions,non-aqueous solutions in common solvents such as alcohols, or solutionsin a liquid oil base.

In some embodiments, the carrier of the composition of the presentinvention may include a release agent such as sustained release ordelayed release carrier. In such embodiments, the carrier can be anymaterial capable of sustained or delayed release of the active toprovide a more efficient administration, e.g., resulting in lessfrequent and/or decreased dosage of the composition, improve ease ofhandling, and extend or delay effects on diseases, disorders,conditions, syndromes, and the like, being treated, prevented orpromoted. Non-limiting examples of such carriers include liposomes,microsponges, microspheres, or microcapsules of natural and syntheticpolymers and the like. Liposomes may be formed from a variety ofphospholipids such as cholesterol, stearylamines orphosphatidylcholines.

The compositions of the present invention may be administeredparenterally in the form of a sterile injectable aqueous or oleaginoussuspension. The term “parenteral” or “parenterally” as used hereinrefers to introduction into the body by way of an injection (i.e.,administration by injection), including, but not limited to, infusiontechniques. The composition of the present invention comprising achemotactic hematopoietic stem cell product is delivered to the subjectby means of a balloon catheter adapted for delivery of the fluidcompositions (i.e., compositions capable of flow) into a selectedanatomical structure.

The sterile composition of the present invention may be a sterilesolution or suspension in a nontoxic parenterally acceptable diluent orsolvent. A solution generally is considered as a homogeneous mixture oftwo or more substances; it is frequently, though not necessarily, aliquid. In a solution, the molecules of the solute (or dissolvedsubstance) are uniformly distributed among those of the solvent. Asuspension is a dispersion (mixture) in which a finely-divided speciesis combined with another species, with the former being so finelydivided and mixed that it doesn't rapidly settle out. In everyday life,the most common suspensions are those of solids in liquid water. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride (saline) solution. Insome embodiments, hypertonic solutions are employed. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For parenteral application, particularly suitablevehicles consist of solutions, preferably oily or aqueous solutions, aswell as suspensions, emulsions, or implants. Aqueous suspensions maycontain substances which increase the viscosity of the suspension andinclude, for example, sodium carboxymethyl cellulose, sorbitol and/ordextran.

Additional compositions of the present invention can be readily preparedusing technology which is known in the art such as described inRemington's Pharmaceutical Sciences, 18th or 19th editions, published bythe Mack Publishing Company of Easton, Pa., which is incorporated hereinby reference.

As used herein the terms “therapeutically effective,” “vascular injuryrepairing amount”, “vascular insufficiency repairing amount”, or“pharmaceutically effective amount” refer to the amount of thecompositions of the invention that result in a therapeutic or beneficialeffect following its administration to a subject. The vascularinsufficiency repairing, vascular injury repairing, therapeutic, orpharmaceutical effect can be curing, minimizing, preventing orameliorating a disease or disorder, or may have any other vascularinsufficiency-reducing, vascular injury-repairing, or pharmaceuticalbeneficial effect. The concentration of the substance is selected so asto exert its vascular insufficiency repairing, vascular injuryrepairing, therapeutic, or pharmaceutical effect, but low enough toavoid significant side effects within the scope and sound judgment ofthe physician. The effective amount of the composition may vary with theage and physical condition of the biological subject being treated, theseverity of the condition, the duration of the treatment, the nature ofconcurrent therapy, the timing of the infusion, the specific compound,composition or other active ingredient employed, the particular carrierutilized, and like factors.

A skilled artisan can determine a pharmaceutically effective amount ofthe inventive compositions by determining the dose in a dosage unit(meaning unit of use) that elicits a given intensity of effect,hereinafter referred to as the “unit dose.” The term “dose-intensityrelationship” refers to the manner in which the intensity of effect inan individual recipient relates to dose. The intensity of effectgenerally designated is 50% of maximum intensity. The corresponding doseis called the 50% effective dose or individual ED50. The use of the term“individual” distinguishes the ED50 based on the intensity of effect asused herein from the median effective dose, also abbreviated ED50,determined from frequency of response data in a population. “Efficacy”as used herein refers to the property of the compositions of the presentinvention to achieve the desired response, and “maximum efficacy” refersto the maximum achievable effect. The amount of the chemotactichematopoietic stem cell product in the pharmaceutical compositions ofthe present invention that will be effective in the treatment of aparticular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. (See, for example, Goodman and Gilman's THE PHARMACOLOGICALBASIS OF THERAPEUTICS, Joel G. Harman, Lee E. Limbird, Eds.; McGrawHill, New York, 2001; THE PHYSICIAN'S DESK REFERENCE, Medical EconomicsCompany, Inc., Oradell, N.J., 1995; and DRUG FACTS AND COMPARISONS,FACTS AND COMPARISONS, INC., St. Louis, Mo., 1993). The precise dose tobe employed in the formulations of the present invention also willdepend on the route of administration and the seriousness of the diseaseor disorder, and should be decided according to the judgment of thepractitioner and each subject's circumstances. It is envisioned thatsubjects may benefit from multiple administrations of the pharmaceuticalcomposition of the present invention.

It is preferred that the pharmaceutical compositions according to thepresent invention contain a minimum number of CD34+ hematopoietic stemcells having a subpopulation of at least 0.5×10⁶ CD34+ cells havingCXCR-4 mediated chemotactic activity per dosage unit for parenteraladministration at the physician's discretion.

In another aspect of the present invention, the pharmaceuticalcompositions of the present invention can further include one or morecompatible active ingredients which are aimed at providing thecomposition with another pharmaceutical effect in addition to thatprovided by the isolated chemotactic hematopoietic stem cell product ofthe present invention. “Compatible” as used herein means that the activeingredients of such a composition are capable of being combined witheach other in such a manner so that there is no interaction that wouldsubstantially reduce the efficacy of each active ingredient or thecomposition under ordinary use conditions. In some embodiments, thecombination therapy comprises administering to a subject in need thereofa pharmaceutical composition comprising a chemotactic hematopoietic stemcell product of the present invention combined with an agent selectedfrom the group consisting of an angiotensin converting enzyme (ACE)inhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, ananti-anginal agent, a vasoactive agent or inotrope, an anticoagulantagent, a fibrinolytic agent, and a hypercholesterolemic agent.

In some embodiments, the composition of the present invention furthercomprises about 0.5% to about 5% albumin. In some embodiments, theminimum amount of albumin is about 0.5%, about 0.75%, about 01.0%, about1.25%, about 1.5%, about 1.75%, about 2.0%, about 2.5%, about 2.75%,about 3.0%, about 3.5%, about 4.0%, about 4.5%, or about 5.0%, expressedas ml/100 cc volume of the composition. In some embodiments, the maximumamount of albumin in the compositions of the present invention is about5.0%, about 4.75%, about 4.5%, about 4.25%, about 4.0%, about 3.75%,about 3.5%, about 3.25%, about 3.0%, about 2.75%, about 2.5%, about2.25%, about 2.0%, about 1.75%, about 1.5%, about 1.25%, or about 1.0%,expressed as ml/100 cc volume of the composition. In some embodiments,the albumin is human albumin. In some embodiments the albumin isrecombinant human albumin.

Methods of the Present Invention

In another aspect, the present invention provides a method of preparingthe pharmaceutical composition comprising a chemotactic hematopoieticstem cell product for treating a subject in need thereof. The methodcomprises the steps of

(1) acquiring a preparation comprising an enriched population of potentCD34+ cells from the subject under sterile conditions by a chemotacticcell acquisition process;

(2) sterilely purifying potent CD34+ cells containing a subpopulation ofpotent cells having chemotactic activity from the preparation;

(3) sterilely formulating the purified potent CD34+ cells to form thechemotactic hematopoietic stem cell product;

(4) sterilely formulating the chemotactic hematopoietic stem cellproduct containing a subpopulation of potent CD34+ cells havingchemotactic activity to form a pharmaceutical composition;

(5) assessing sterility of the pharmaceutical composition;

(6) releasing the sterile pharmaceutical composition as eligible forinfusion into the subject;

(7) loading a therapeutically effective amount of the pharmaceuticalcomposition into an intravascular delivery apparatus; and

(8) optionally transporting the delivery apparatus containing thetherapeutically effective amount of the sterile pharmaceuticalcomposition comprising the chemotactic hematopoietic stem cell productto a cardiac catheterization facility for infusion in to the subject.

In one embodiment, step (2) is initiated within about 12 hours to about24 hours of completion of acquiring step (1). In another embodiment,releasing step (7) proceeds only if the sterile formulated cell productis to be infused into the subject within about 48 hours to about 72hours of completion of acquiring step (1). In another embodiment, step(2) is initiated within about 12 hours to about 24 hours of completionof acquiring step (1), and releasing step (6) proceeds only if thesterile formulated cell product is to be infused into the subject withinabout 48 hours to about 72 hours of completion of acquiring step (1).

In one embodiment, step (5), i.e., the step of assessing sterility ofthe pharmaceutical composition further comprises the steps of (i)centrifuging the chemotactic hematopoietic stem cell product comprisingpotent CD34+ cells to form a cell pellet and a supernatant, the cellpellet comprising the potent CD34+ cells; (ii) sterilely removing thesupernatant without disturbing the cell pellet; and (iii) analyzingwhether the supernatant is contaminated by a microbe thereby determiningthe sterility of the cell pellet.

In one embodiment, in step (a), the chemotactic cell acquisition processis a mini-bone marrow harvest technique used to acquire a preparationcomprising an enriched population of potent CD34+ cells from the bonemarrow of the subject under sterile conditions. For the bone marrowharvest technique, step (a) of the method further comprises the steps:(i) preloading harvesting syringes with heparin prior to harvesting bonemarrow from a subject; (ii) aspirating the bone marrow from a leftposterior iliac crest and a right posterior iliac crest of the subjectusing the harvesting syringes and a mini-bone marrow harvest techniqueto form harvested bone marrow; and (iii) infusing the harvested bonemarrow into a collecting bag. In one embodiment, the harvesting syringesin step (i) and the collecting bag in step (iii) contain a preservativefree heparinized solution comprising 0.9% normal saline. The finalconcentration of heparin in the heparinized saline solution is about 20units per ml to about 25 units per ml.

Optionally, in one embodiment of the method, the harvested bone marrowis transported to a processing facility different from the facility fromwhich the bone marrow was harvested. In one embodiment, the method fortransporting the harvested bone marrow to the processing facilitycomprises the steps (a) placing the harvested bone marrow in acollection bag; (b) placing the collection bag in a secondary bag; (c)placing the secondary bag containing the collection bag in a shippingcontainer comprising an interior compartment containing frozen wet iceand at least one sheet of bubble wrap; (d) affixing a temperature tagmonitor to the interior compartment of the shipping container; (e)sealing the shipping container; and (f) shipping the shipping containerto the processing facility.

In another aspect, the present invention provides a method of treatinginjury due to vascular insufficiency in a subject in need thereof, themethod comprising the steps: (a) evaluating whether the subjectqualifies for therapy with the pharmaceutical composition of the presentinvention; (b) preparing the pharmaceutical composition comprising achemotactic hematopoietic stem cell product; (c) loading thepharmaceutical composition into an intravascular delivery apparatus; (d)delivering a therapeutically effective amount of the pharmaceuticalcomposition to the subject intravascularly (meaning inside a bloodvessel); and (e) monitoring the subject's cardiac function.

According to one embodiment of the present invention, the subject inneed thereof is a revascularized myocardial infarction patient. The term“revascularized” as used in this embodiment refers to the successfulplacement of a stent. Clinical evaluations, for example, of coronaryinsufficiency using non-laboratory tests, cardiac catheterization,measurement of inflammatory cytokines, and measurement of cardiacbiomarkers can be used to determine the appropriate time to administerthe pharmaceutical compositions in accordance with the methods of thepresent invention. In some embodiments, detection of peak inflammatorycytokine cascade production enables the administration to be tailored atthe window most crucial for the particular subject. In some embodiments,peak inflammatory cytokine cascade production is determined by themeasuring the levels of the appropriate cytokine(s) in the plasma and orurine. In other embodiments, the level(s) of the appropriate cytokine(s)is/are measured immunochemically, for example, by a sandwich enzymeimmunoassay, by enzyme-linked immunosorbent assays (ELISA) or bymultiplex bead kits.

According to one embodiment, the composition is administered to thesubject after an inflammatory cytokine cascade production peaks. In someembodiments, the composition is administered to the revascularizedmyocardial infarction patient about 5 days to about 14 dayspost-infarction. The minimum time in which to administer the compositionto the revascularized myocardial infarction patient is about 5, 6, 7, 8,9, 10, 11, 12, 13, or 14 days. The maximum time in which to administerthe composition is about 14, 12, 11, 10, 9, 8, 7, 6, or 5 days.

The intravascular delivery apparatus used to deliver the pharmaceuticalcomposition of the present invention to a subject in need thereofcomprises an infusion syringe, a flushing syringe, a four-way stopcock,and a balloon catheter. In one embodiment, the intravascular deliverycomprises (a) an infusion device attached to a sterile four-way stopcockcontaining the pharmaceutical composition comprising the chemotactichematopoietic stem cell product; (b) a flushing device attached to thesterile four-way stopcock, the flushing device containing a flushingsolution, and (c) a catheter attached to the delivery apparatus by thesterile four-way stopcock. According to one embodiment, the infusiondevice is a syringe made of any suitable material. The body and handleof suitable four way stopcocks may be made of the same or a differentmaterial. Examples of suitable four-way stopcocks includes, withoutlimitation, a stopcock having a polycarbonate body/polycarbonate handle,a stopcock having a polyethylene body/polyethylene handle, a stopcockhaving a polycarbonate body/polyethylene handle, or a disposablestopcock. In another embodiment, a device is further attached to thestopcock to regulate the pressure exerted on the delivered solution. Insome embodiments an integral flush device or syringe is attached to thestopcock. In one embodiment, the catheter is a balloon catheter. Theterm “balloon catheter” refers to a type of “soft” thin flexible tubehaving an inflatable “balloon” at its tip which is used during acatheterization procedure to enlarge a narrow opening or passage withinthe body. The deflated balloon catheter is positioned, inflated toperform the necessary procedure, and deflated again to be removed.

The viability and potential efficacy of the chemotactic hematopoieticstem cell product of the present invention comprising potent CD34+ cellsdepends on the cells maintaining their potency as they pass through acatheter. The catheter used in the methods of the present invention hasan internal diameter of at least 0.36 mm. Any type of catheter having aninternal diameter of at least 0.36 mm may be effective in delivering thepharmaceutical compositions of the present invention.

For example, a flow control catheter, which slows drainage of bloodthrough the coronary artery vasculature allows the cells time to transitthrough the blood vessel wall and into the tissue.

In some embodiments, the catheter is a balloon catheter. For example,without limitation, the following balloon dilatation catheters availablefrom Cordis, Boston Scientific, Medtronic and Guidant having an internaldiameter of about 0.36 mm have been validated (see Table 1).

TABLE 1 Balloon catheter validated for infusion of selected CD34+ cellsthrough the IRA Manu- Name and Model Balloon Lumen Internal facturer No.Dimensions Diameter Cordis Raptor OTW 15 mm × 3.0 mm 0.36 mm (0.14 in.)579-130 Boston OTW Maverick 15 mm × 3.0 mm 0.36 mm (0.14 in.) Scientific20620-1530 Medtronic OTW Sprinter SPR 15 mm × 3.0 mm 0.36 mm (0.14 in.)3015W Guidant Voyager OTW 15 mm × 3.0 mm 0.36 mm (0.14 in.) 1009443-15

In addition, catheters have been described having a fluid delivery portadjacent the balloon such that the balloon may be inflated against avessel wall to isolate the delivery site from hemodynamics opposite theballoon from the port, which may be located distally of the balloon.Additionally, balloon catheters have been disclosed having lumens endingin side ports disposed proximally to the balloon catheter; these ballooncatheters generally may be referred to as “balloon/delivery” catheters,although particular references may use different descriptors. See, e.g.,U.S. Pat. No. 5,415,636 to Forman, incorporated by reference.

In some embodiments, the method of treating or repairing a vascularinjury comprises administering the composition via ballooncatheterization into an infarcted artery. In some embodiments, followingangioplasty a delivery balloon catheter is inserted via a femoral arteryinto a desired coronary artery, such as the left anterior descendingcoronary artery. Some medical conditions may require both a ballooncatheter and a fluid delivery catheter to facilitate treatment.

In some embodiments, a catheter is used to directly inject cells intothe myocardium.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges also is encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

As used herein and in the appended claims, the singular forms “a”,“and”, and “the” include plural referents unless the context clearlydictates otherwise. All technical and scientific terms used herein havethe same meaning.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be confirmed independently.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Selection of Eligible Subjects

Subjects/patients presenting with symptoms and clinical findingssuggestive of a myocardial infarction will receive emergency diagnosticand clinical management according to institutional guidelines. If atransmural (meaning through the wall) myocardial infarction isconfirmed, the time of first symptoms and the time of successful stentplacement will be recorded. Revascularized subjects will receiveappropriate medical management to reduce ventricular wall stressesaccording to institutional guidelines. The term “revascularized” as usedin this embodiment, refers to the successful placement of a stent.

All types of stents, including drug-eluting stents (e.g., paclitaxel orsirolimus) are acceptable for use in the revascularization of theinfarct related artery (“IRA”). Previous studies employing ballooncatheters to infuse cell products have reported no limits for referencevessel diameter for the placement of the stent. Since this study isdesigned to distribute the cell product into the IRA circulation, and inan attempt to limit the potential for damage to very small vessels, thepresent invention requires that stents be placed prior to infusion ofthe chemotactic hematopoietic stem cell product of the presentinvention.

Stent-related drug effects occur predominantly at the site of contact ofthe stent with the vessel wall. Consequent to balloon dilatation, thereis limited blood flow across the stent during cell infusion, andtherefore no significant adverse drug-mediated effect on the CD34⁺ cellsin the chemotactic hematopoietic stem cell product is expected.Moreover, prior clinical studies have shown that by 96 hours afterdrug-eluting stent placement, whole blood levels of either paclitaxel orsirolimus are below the limits of detection. Therefore, tissue levels inthe myocardial sites to which the infused CD34⁺ cells havingCXCR-4-mediated chemotactic activity are intended to migrate areexpected to be inconsequential. See Sousa, J. et al., Circulation 107:2274-79, 2383-89 (2003).

During revascularization, a subject's cardiac function and perfusionwill be assessed by standard methods. Relevant measures of cardiacfunction following a myocardial infarction include assessment of globaland regional ejection fraction, ventricular volumes, resting and stressperfusion, segmented wall motion and infarct size.

Echocardiography, radionuclide scanning (e.g., Multiple GatedAcquisition scan (MUGA), a nuclear scan that evaluates the pumpingfunction of the ventricles, chambers and how the heart contracts) andleft ventriculography all are readily available and accurate measures ofleft ventricular ejection fraction (“LVEF”). Echocardiography has beenutilized to determine end-systolic and end-diastolic volumes by usingthe biplane area length method. The left ventricular wall motion scoreindex (“WMSI”) is calculated by dividing the left ventricle intosegments and then grading systolic wall motion and thickening by asemi-quantitative score index as follows: 1=normal or hyperkinesia;2=hypokinesia; 3=akinesia; and 4=dyskinesia. As used herein, the term“hyperkinesias” refers to excessive movement or muscular activity; theterm “hypokinesia” refers to diminished or slow movement or muscularactivity; the term “akinesia” refers to the absence or loss of movementor muscle activity; and the term “dyskinesia” refers to abnormalmovement or muscular activity. The WMSI then is obtained by dividing thesum of the segment scores by the number of segments assessed. SeeSchiller, N. B. et al., J. Am. Soc'y Echocardiogr. 2: 358-67 (1989).Newer echocardiographic modalities allow for assessment of regionalejection fraction, myocardial perfusion, and post stress wallabnormalities.

Other measures of cardiac function in the post-infarct period includeassessment of the stroke volume index and velocity of circumferentialfiber shortening. Strauer, et al., Circulation 106: 1913-18 (2002).Assessment of repair of infarcted myocardium also has includedevaluation of peri-infarct region perfusion using thallium scintigraphy.Id. Most recently, magnetic resonance imaging (MRI) appears to be themost useful tool for assessing cardiac function and viability (infarctsize) in this setting. See Yin, A, et al., Blood 90: 5002-5012 (1997).

The day after successful stenting, subjects will be assessed for studyeligibility and, if appropriate, will be offered informed consent toparticipate in the study. Subjects exhibiting symptoms for no more thanthree (3) days prior to successful stent placement will be assessed,prior to discharge, for study eligibility. Subjects found to meeteligibility criteria (see infra) will be offered informed consent toparticipate.

Consented subjects will have a study entry echocardiogram no sooner than96 hours after stent placement. Subjects are eligible to proceed onstudy if the LVEF is less than or equal to 50% on echocardiography and asegmental ventricular wall abnormality is observed in the IRA. Eligiblesubjects immediately can complete baseline cardiac function andperfusion assessment.

Specifically, baseline cardiac function includes: transthoracicechocardiography at rest and with low dose dobutamine to assess cardiacfunction including ejection fraction, end systolic and diastolicvolumes, and wall motion score index and viability. Myocardial contrastechocardiography will be used to assess segment wall motion andmyocardial blood flow at the tissue level. Myocardial strain rates alsowill be assessed. Perfusion will be assessed using a routine Tc-99mSestamibi radionuclide scan at rest and after intravenous adenosine.Regional and global wall motion, infarct size, and left ventricular(“LV”) volumes will be measured using MRI. Subjects will receiveGadolinium contrast during scanning. MRI scan will use the breathholding technique. Steady state precession imaging to obtain global andregional LV function will be performed as will Gadolinium imaging. Leftventricular end systolic and diastolic volumes, LVEF, LV end diastolicdimension, wall thickness in systole and diastole of the infarctedregion, and infarct size will be reported using the AHA/AVV 17-segmentmodel with transmural extent of the infarct reported as <25%, 26%-50%,51%-75% and >76%. A core review laboratory will assess MRI with theinterpreter blinded to the study cohort.

To be selected for this study, subjects must meet all of the followingclinical criteria (“inclusion criteria”):

-   -   Age: 18-75 years;    -   Acute ST segment elevation myocardial infarction meeting ACC/AHA        criteria, with symptoms of chest pain within 3 days of        admission. Criteria include (ST elevation >1 mm in limb leads or        2 mm in two or more precordial leads and increased levels of        troponin, creatine kinase MB (CPK MB) or both), New York Heart        Association (NYHA) heart failure class (to be recorded) of I, II        or III;    -   Eligible for percutaneous coronary intervention (PCI);    -   Eligible for MRI;    -   Eligible for Single Proton Emission Computed Tomography (SPECT)        imaging;    -   Echocardiograph lab conclusion of ability to adequately assess        cardiac parameters after review of admission echocardiography;    -   Study entry echocardiogram (96 to 144 hours {i.e., about 4 days        to about 6 days} after stent placement), LVEF less than or equal        to 50% on echocardiography, and segmental ventricular wall        abnormality in the IRA circulation by echocardiography after        reperfusion;    -   Subject must be able to provide informed written consent and        must be willing to participate in all required study follow-up        assessments;    -   Subjects must have a hemoglobin content (Hgb) >10 grams/dL,        white blood cell count (WBC) >3500 cells/mm³, a platelet        count >100,000 cells/mm³ and a international normalized ratio        (INR, a blood coagulation test) <2.0 the day before the bone        marrow collection;    -   Subjects must have a serum creatinine <2.5, total bilirubin <2.0        within 7 days of the bone marrow collection:    -   IRA and target lesion must be clearly identifiable when disease        is present in more than one vessel;    -   Successful reperfusion and intracoronary stent placement, with        Thrombolysis In Myocardial Infarction (TIMI) 2 or 3 flow and IRA        with <20% stenosis after revascularization;    -   Subjects must be deemed eligible to receive conscious sedation,        mini-bone marrow harvest, and second catheterization for        Chemotactic hematopoietic stem cell product infusion;    -   The type of stent used and time and date inserted must be        recorded;    -   Drug eluting stents should be limited to paclitaxel or sirolimus        types;    -   Included subjects must have an expected survival of at least one        year and must not have multiple vessel disease after        revascularization, or be expected to require intervention within        6 months of study entry.

Subjects who satisfy any one of the following criteria do not qualifyfor, and will be excluded from, the study (“exclusion criteria”):

-   -   Subjects who are not candidates for percutaneous intervention,        conscious sedation, MRI, SPECT imaging or mini-bone marrow        harvest;    -   History of sustained chest pain unrelieved by nitrates,        occurring 4 or more days before revascularization;    -   Subjects who fail to re-perfuse the infarct related coronary        artery or to have successful stent placement;    -   Echocardiography lab conclusion after admission echocardiography        review that study is not adequate to assess cardiac parameters;    -   Subjects presenting with cardiogenic shock (systolic pressure        <80 on vasopressors or intra aortic counterpulsation);    -   Subjects with a side branch of the target lesion >2 mm and with        ostial narrowing >50% diameter stenosis after revascularization;    -   Subjects unable to receive aspirin, clopidogrel or ticlopidine;    -   Subjects receiving warfarin must have an INR less than or equal        to 2; the term INR refers to INR International Normalized Ratio,        which is a system established by the World Health Organization        (WHO) and the International Committee on Thrombosis and        Hemostasis for reporting the results of blood coagulation        (clotting) tests;    -   Subjects with severe aortic stenosis;    -   Subjects with severe immunodeficiency states (e.g., AIDS);    -   Subjects with cirrhosis requiring active medical management;    -   Subjects with malignancy requiring active treatment (except        basal cell skin cancer);    -   Subjects with documented active alcohol and/or other substance        abuse;    -   Females of child bearing potential unless a pregnancy test is        negative within 7 days of the mini-bone marrow harvest;    -   Subjects with ejection fractions greater than 50% on study entry        echocardiogram (96 to 144 hours after stent placement);    -   Subjects with less than three months of planned anti-platelet        therapy post index procedure;    -   Subjects with multi vessel disease after revascularization        requiring subsequent planned intervention during the next 6        months;    -   Subjects with participation in an ongoing investigational trial;    -   Subjects with active bacterial infection requiring systemic        antibiotics.

Baseline assessments of cardiac function and cardiac perfusion will beobtained one day prior to the planned mini-bone marrow harvest andinfusion of the chemotactic hematopoietic stem cell product (see infra).A mini-bone marrow harvest (“MMH”) will be performed the day followingbaseline assessment of cardiac function and cardiac perfusion.

Example 2 Cardiac Catheterization

Sterile Preparation and Draping

The subject will be brought into the Cardiac Catheterization Laboratoryafter the investigator has obtained an informed consent. The subjectwill receive a sterile preparation and draping in the CardiacCatheterization Laboratory.

Cardiac Catheterization

Vascular access will be obtained by standard technique using right orleft groin. A sheath will be placed in the femoral artery or the rightor left brachial artery. Coronary arteriographic examination will beperformed by obtaining standard views of both right and left coronaryarteries. Multiple views will be obtained to identify the previouslystented infarct related artery. All subjects will receive standardmedications during the catheterization procedure in accordance withroutine practice.

Example 3 Acquisition Process for Acquiring Chemotactic HematopoieticStem Cell Product that can then be Enriched for CD34+ Cells

While it is contemplated that any acquisition process appropriate foracquiring the chemotactic hematopoietic stem cell product comprisingpotent CD34+ cells is within the scope of the present invention, thefollowing example illustrates one such process referred to herein as amini-bone marrow harvest technique.

Preparation of Harvesting Syringes

Prior to the bone marrow harvest, forty 10 cc syringes loaded with about2-ml of a preservative free heparinized saline solution (about 100units/ml to about 125 units/ml, APP Cat. No. 42592B or equivalent) willbe prepared under sterile conditions. Heparin will be injected via asterile port into each of two 100-ml bags of sterile 0.9% normal salinesolution (“Normal Saline”, Hospira Cat. No. 7983-09 or equivalent)following removal of 10 cc to 12.5 cc of normal saline from each bag,resulting in a final heparin concentration of about 100 units/ml (U/ml)to about 125 units/ml (U/ml). 2-ml of the preservative free heparinsolution (about 100 U/ml to about 125 U/ml) will be loaded under sterileconditions into each of the forty 10 cc syringes, which then are cappedand placed into a sterile bag for transport to the harvesting site.

Subjects will be prepared for bone marrow harvest after written informedconsent is obtained as detailed in Example 1. Conscious sedation will beprovided using standard institutional procedures and guidelines. Bonemarrow harvest will be conducted under sterile conditions. The term“sterile conditions” as used herein includes proper scrubbing andgowning with a sterile mask and gloves worn by the harvesting attendingand assistant. The harvesting procedure can be performed outside of anoperating room as follows: after sterile prepping and draping, eachiliac crest should be anaesthetized with a 1% lidocaine solution using aminimum of 10-ml for each crest. The area of anesthesia should be acircular area no less than 10 cm in diameter. The harvesting needle isinserted until the iliac crest is punctured. The cap and stylet isremoved and 2-ml of marrow is harvested into the 10-ml harvestingsyringe containing 2-ml of the heparin solution. The syringe then isremoved and placed on the sterile field. After re-inserting the stylet,the harvesting needle is advanced slightly and then rotated 90°. Thestylet is then removed and an additional 2-ml of marrow is drawn intothe harvesting syringe retrieved from the sterile field. This procedureis repeated two more times until the harvesting syringe contains 8-ml ofmarrow for a total of 10-ml of heparinized marrow at a final heparinconcentration of about 20 U/ml to about 25 U/ml. Finally the fullharvesting syringe is handed to the harvesting assistant and shaken andinfused in the sterile collecting bag as described below. The harvestingphysician then takes the other harvesting needle that had been flushedpreviously with the heparin solution and repeats this process.

The full harvesting syringe is infused in the sterile collecting bag asfollows. The harvesting assistant is handed the full harvesting syringeand empties it in the 500-ml collecting bag though the sterile adaptorattached to the bag. Then the harvesting needle is flushed with theheparin solution in the flushing syringe and retuned to the sterilefield.

The harvesting process is repeated on one iliac crest until about 19syringes have been collected and emptied in the collecting bag. The sameprocess is repeated on the other iliac crest until another about 19syringes have been filled. A total of thirty-eight 8 ml aspirations fromboth iliac crest (ideally 19 from each iliac crest) will result in302-ml of bone marrow harvested in a final volume of 380 ml at a heparinconcentration of about 20 U/ml to about 25 U/ml.

The collecting bag is sealed by tying off the connecting tube threetimes and then clamped distal to the ties. The bag is appropriatelylabeled “Human Bone Marrow Collection” and the results of the harvestingprocedure, including final volume collected and any procedure relatedcomplication, are recorded on the Mayo Clinical Risk Score (MCRS) casereport form. The completed label is affixed to the bone marrow bag. Thebag then is placed in a sterile carrying bag to be transported to theprocessing facility.

Example 4 Preparation of the Bone Marrow Product for Transportation

In one embodiment, the harvested bone marrow is transported to theprocessing facility as follows. When the clinical site is prepared toship the bone marrow preparation, 24-hour notice will be provided to theprocessing facility. The processing laboratory will make shippingarrangements at the earliest possible time for pickup for same daydelivery to the processing laboratory. Immediately after the bone marrowis collected, the bone marrow product will be placed in the suppliedshipping container. The shipping container contains two small blocks offrozen wet ice on the bottom and a sheet of bubble wrap on top of thewet ice. The bone marrow product is placed into a secondary bag and thesecondary bag is placed on top of the bubble wrap. A temperature tagmonitor (a sensor used to monitor the internal temperature) is affixedto the interior of the box. Another layer of bubble wrap then is placedon top of the product before the shipping container is sealed off.

Example 5 Selection of CD34+ Cells from the Harvested Bone MarrowProduct

CD34⁺ cells will be isolated from the harvested bone marrow product. Inone embodiment, CD34⁺ cells will be isolated using the anti-CD34monoclonal antibody (Mab), Dynabeads® M-450 Sheep anti-Mouse IgG, andPR34+™ Stem Cell Releasing Agent components of the Isolex 300i MagneticCell Selection System (Baxter Healthcare Corp. Cat. No. 4R9734) asdescribed in U.S. Pat. Nos. 5,536,475, 5,035,994, 5,130,144, 4,965,204,5,968,753, 6,017,719, 6,251,295, 5,980,887, 6,676,937, U.S. PublishedApplication No. 2003/0232050, and the Isolex 300i Package Insert, eachof which is incorporated herein by reference. This operating system hasbeen adapted for isolation of CD34⁺ cells from bone marrow according tothe present invention.

Upon arrival at the processing laboratory, the harvested bone marrowproduct (in the collecting bag) is inspected immediately and the bagchecked for any leakage. The collection should be free flowing with noapparent clumps and should not be hemolyzed. The collection will not beused if the integrity of the bag has been breached in any way.

The bone marrow product should be processed within about 12 hours toabout 24 hours of inspection. A 300-ml or 400-ml transfer pack containeris obtained, and a plasma transfer set is attached to the sampling portof the container. The bone marrow product is transferred from thecollecting bag to the transfer pack container. The pooled bone marrowcollection product is mixed thoroughly by inverting the container twenty(20) times.

The pooled bone marrow collection product then is sampled for analysis.In one embodiment, a total volume of 2.0 ml of the product is removedand aliquoted as follows: 0.3 ml is used for a duplicate run of CompleteBlood Count (CBC) using a hematology analyzer; 0.2-ml is dispensed intoa 75×100-mm glass tube for the detection of Gram positive and Gramnegative bacteria by Gram Stain (Gram Stain Kit, VWR, Cat. NO.BB231401); as a sterility check, 0.6-ml is dispensed into a Tryptic SoyBroth (TSB) (VWR, Cat. No. 29446-184) bottle for aerobic bacteria growthassay, 0.6-ml is dispensed into a Fluid Thioglycollate Media (FTM) (VWRCat. # 29446-138) bottle for anaerobic bacteria growth assay, and 0.3-mlis used in flow analysis for CD34⁺ cell enumeration and cell viability.

The collection is weighed on an electronic scale, and the appropriatetare weight of the collection bag recorded. The relationship of thevolume of the bone marrow product to the weight of the product can beexpressed as

Volume (ml)=[Weight (gm) of product−Tare weight of bag (gm)]÷1.06(gm/ml)  (Formula 1)

The number of Total Nucleated Cells (TNC) in the bone marrow product iscalculated using the white blood cell (WBC) count obtained from the CBCaccording to the following relationship:

TNC=WBC/μl×1000×Product volume (ml)  (Formula 2)

The number of CD34+ cells in the bone marrow product is calculated fromthe following relationship:

Total CD34⁺ cells in the bone marrow product=Number of CD34⁺cell/μl×1,000×Product volume (ml)  (Formula 3)

The Red Blood Cell (RBC) volume of the bone marrow collection product iscalculated from the following relationship:

RBC volume (ml)=Product volume (ml)×Hematocrit (%)/100  (Formula 4),

If the collection contains more than 20 ml of RBC, red blood celldepletion is required. RBCs are depleted by centrifugation.Centrifugation at 1000×g for 20 minutes at ambient temperature isperformed to separate the buffy coat from the RBCs. The term “buffycoat” refers to a thin grayish white fraction of a blood sample thatcontains most of the white blood cells (leukocytes). Immediately aftercentrifugation, a 60 ml syringe is connected to the bottom of thecentrifugation bag and the RBCs are removed. More than one syringe maybe needed to collect all the packed RBC. The RBC depleted bone marrowproduct then is washed to remove fat contents.

A 1-ml syringe is used to remove 0.3-ml of the RBC-depleted bone marrowcell product through the transfer set attached to the product bag and aCBC performed. The TNC of the RBC depleted bone marrow product isdetermined from the relationship:

Total TNC of the RBC depleted product=WBC/μl of RBC depletedproduct×1000×180-ml  (Formula 5)

The TNC recovery of the RBC depleted product, which must be at least 80%of the original product count, is calculated from the relationship:

TNC recovery=TNC of the RBC depleted product÷TNC of the unprocessedproduct×100%  (Formula 6)

The total RBC volume is calculated as described supra; the RBC volume inthe RBC depleted product should be less than <20-ml.

In one embodiment according to the present invention, the Isolex 300isystem is used to process the RBC-depleted product or the bone marrowproduct whose RBC volume is <20 ml according to the following processingsteps:

(i) The bone marrow is washed automatically to remove platelets;

(ii) CD34 positive (CD34+) cells are labeled specifically for selectionby incubation with the Isolex 300i CD34 monoclonal antibody (Mab);

(iii) Unbound reagent is removed by washing the cell suspension withbuffer solution;

(iv) Sensitized CD34+ cells (meaning CD34+ cells labeled with CD34 Mab)are captured by Dynabeads M-450 Sheep anti-Mouse IgG;

(v) A selection column is used to separate the magnetically-labeledDynabeads having captured CD34+ cells from unwanted cells, which arewashed through the selection column and collected in the NegativeFraction Bag; and

(vi) PR34+ Stem Cell Releasing Agent releases CD34+ cells from thecolumn, and the CD34+ cells are collected in the End Product Bag. Thesystem performs several washing steps, disposing of most of the liquidinto the Buffer Waste Bag.

The Isolex® selected CD34+ fraction is assayed as follows to determineWBC and CD34+ cell yields. The volume of the CD34 Positive Fraction isdetermined by mixing the cells in the End Product Bag; the bag is gentlymassaged by hand to ensure even cell distribution. A transfer set isinserted into the sampling port of the End Product Bag and a 60-mlsyringe attached. The cell suspension is withdrawn into the syringe(maximum 50-ml at a time) in order to measure the total volume.

A 3-ml or 5-ml syringe is used to remove a 2.0-ml sample from the EndProduct Bag through the transfer set for quality control testing. Thealiquoted volumes of the samples and the analyses performed on thosesamples are as previously described, i.e., CBC: 0.3-ml; Gram stain:0.3-ml; CD34+ cell enumeration and cell viability: 0.2-ml.

The total TNC of the CD34 Positive Fraction is calculated from therelationship:

Total TNC of the Positive Fraction=WBC/μl of the PositiveFraction×1000×Volume of the Positive Fraction  (Formula 7)

The TNC recovery of the Positive Fraction, which must be less than 5% ofthe original product count, is calculated from the followingrelationship:

TNC recovery=Total TNC of the Positive Fraction÷Total TNC of theunprocessed product×100%  (Formula 8)

The total number of viable CD34+ cells in the Positive Fraction isdetermined from the following relationship:

Total CD34+ cells in the Positive Fraction=Number of CD34+ cells/μl ofthe final product×1,000×Final product volume (ml)  (Formula 9)

The CD34+ cell recovery of the Positive Fraction is calculated from thefollowing relationship:

CD34+ cell recovery=Total CD34+ cells of the Positive Fraction÷TotalCD34+ cells of the unprocessed product×100%  (Formula 10).

Example 6 Preparation of Selected CD34+ Cells for Transfusion

Samples of the chemotactic hematopoietic stem cell product will beremoved to be assayed for WBC count, by flow cytometry (for CD34+ cellenumeration and viability), Gram stain, and sterility.

CD34+ cells are characterized by flow cytometric analysis featuringCD34^(bright) and CD45^(dim) fluorescence by double labeling withanti-CD34 and anti-CD45 antibodies (Beckman Coulter, PN IM3630). CD34+cells and CD45⁺ cell viability is determined by excluding the dyingcells which take up the intercalating DNA dye 7-aminoactinomycin D(7AAD). See Brocklebank A M, Sparrow R L. Cytometry. 2001; 46:254-261(2001); Barnett D, et al. Br. J Haematol. 106:1059-1062 (1999);Sutherland, et al., J Hematotherapy 5:213-226 (1996), and U.S. Pat. Nos.4,520,110; 4,859,582; 5,055,556; European Patent No. 76.695; CanadianPatent No. 1,179,942 (PE, APC); U.S. Pat. No. 4,876,190 (PerCP); U.S.Pat. Nos. 5,268,486; 5,486,616; 5,569,587; 5,569,766; 5,627,027 (Cy);U.S. Pat. Nos. 4,714,680; 4,965,204; 5,035,994 (CD34); U.S. Pat. No.5,776,709 (Lyse/no-wash method); U.S. Pat. Nos. 5,723,218 and 5,187,288(TruCOUNT Tubes), the contents of each of which is incorporated byreference herein in its entirety.

Any flow cytometer or an equivalent device can be used for conductinganalysis of CD34+ cell enumeration and viability. In one embodiment, theprocessing laboratory employs a BD FACSCalibur™ flow cytometer and BDFACSComp™ software is used for instrument setup and monitoring. Atemplate and a panel of legend labels are preinstalled for acquisitionand analysis. Prior to use, the reagents, namely CD45FITC/CD34PE,Stem-Count Fluorospheres, Concentrated Ammonium Chloride LysingSolution, and 7AAD Viability Dye, are brought to ambient temperature.CD34+ cell controls are run as a positive control to affirm that theinstrument is set up for analyzing CD34+ cells, and the results arecompared with the manufacturer's pre-determined CD34 percent range.

The unprocessed bone marrow product and Isolex processed chemotactichematopoietic stem cell products may be analyzed by many differentprocedures. In one embodiment, or example, immediately upon receivingthe sample, if the WBC count of the sample is greater than 2×10⁷ cellsper ml, the sample is diluted with Sheath fluid to achieve a cell countof about 2×10⁷ WBC per ml. 100 μl of the diluted product is aliquotedinto two 15×100 mm tubes. Using a micropipetter, 201 of CD45FITC/CD34 PEand 7-AAD viability dye reagent are added into each tube and the samplesgently vortexed. The tubes are covered with aluminum foil and left atambient temperature for 15 to 20 minutes. RBCs are lysed by adding 1.5ml of 1× Lysing Solution to each tube, vortexing gently. The tubes areincubated for ten minutes at ambient temperature, protected from light.The samples are stored at about 2° C.-about 8° C. (i.e., on an ice bath)protected from light until data acquisition is performed. Dataacquisition must be performed within one hour of adding the lysingbuffer. Before data acquisition, Stem-Count Fluorospheres areresuspended by end-over-end rotation (10 times). 100 μl of Fluorospheresis added to each tube and gently vortexed taking care not to generateair bubbles. The absolute count of CD34+ cells in the product iscalculated from the relationship:

$\begin{matrix}{{{{Number}\mspace{14mu} {of}\mspace{14mu} {viable}\mspace{14mu} {CD}\; 34} + {{cells}\mspace{14mu} {per}\mspace{14mu} {µl}\mspace{14mu} {of}\mspace{20mu} {product}}} = \frac{{LCD}\; 34 \times {FAC}}{F}} & \left( {{Formula}\mspace{14mu} 11} \right)\end{matrix}$

where LCD34 is the averaged number of events for Live CD34⁺/All CD 45⁺;“FAC” is Fluorospheres Assayed Concentration; and F is the averagednumber of Fluorosphere singlets counted.

The volume of CD34+ Positive Fraction is calculated to obtain the numberof CD34+ cells required for the required dosing. The Required PositiveFraction Volume (ml) is defined as:

The Requested CD34+ cell dosage÷(Total CD34+ cells per μl in thePositive Fraction×1,000).  (Formula 12)

An appropriate number of cells is dispensed into a 50 ml conical tubeand centrifuged at 500×g for 10 minutes. The supernatant is removedusing a 30 ml serological pipette and disposed of as waste whileexercising care not to disperse the cell pellets at the bottom of thetubes during this process. The infusion solution (20 ml) is added intothe CD34+ Cell Positive Fraction tube and the cells dispersed using a 10ml serological pipette by repeat pipetting. The resuspended cells arecentrifuged for 10 minutes at 500 g. A 30 ml serological pipette is used(without disturbing the cell pellet) to transfer thesupernatant/infusion solution into a 50 ml conical tube with a label“Positive Fraction Supernatant” affixed. The tube containing thesupernatant is vortexed to homogenize the solution. A 10 ml serologicalpipette is used to transfer 10 ml of the homogenized supernatant back tothe CD34+ Cell Positive Fraction tube. The remaining 10 ml of suspensionin the Supernatant tube will be used for sterility testing (5 ml eachinto a TSB (Trypticase Soy Broth) bottle and an FTM (FluidThioglycollate) bottle). The cells in the CD34+ Cell Positive Fractionare resuspended by slowly withdrawing and aspirating through a blunt endneedle affixed to a 10 ml syringe (Infusion Syringe) several times. Thecell suspension is withdrawn into the syringe, any air bubbles areaspirated off, and the blunt end needle removed. The infusion syringe isattached to the injection port of a 4-way stopcock.

The chemotactic hematopoietic stem cell product of the present inventionwill be released for infusion only if it meets the following criteria:

-   -   CD34+ cell purity of at least about 70%, 75%, 80%, 85%, 90% or        95%;    -   A negative Gram stain result for the selected positive fraction;    -   Endotoxin Levels: less than about 0.5 endotoxin units/ml;    -   Viable CD34+ cell yield of the “Chemotactic hematopoietic stem        cell product” meets the required dosing as per the treatment        cohort;    -   CD34+ cells are at least about 70%, 75%, 80%, 85%, 90% or 95%        viable by 7-AAD;    -   USP sterility result for “Positive Fraction Supernatant”:        negative (14 days later); and    -   Bone marrow CD34⁺ cell selection was initiated within about 12        hours to about 24 hours of completion of bone marrow harvest.

Sterility assessment on the stem cell product including gram stainingand endotoxin will be performed prior to product release for infusion.USP sterility (bacterial and fungal) culture will be performed and theresults will be reported to the principal investigator. In the event ofa positive USP sterility result, the subject and attending physician oncall will be notified immediately, provided with identification andsensitivity of the organism when available, and documentation ofappropriate anti-microbial treatment and treatment outcome will berecorded by the investigative site and the sponsor.

After meeting these release criteria, the chemotactic hematopoietic stemcell product will be released for infusion and packaged fortransportation to the catheterization facility. A sample also will besent for in vitro testing. Product will be released only if CD34+ cellselection is initiated within 12 hours to about 24 hours of completionof bone marrow harvest and only if it is to be infused within about 48hours to about 72 hours of completion of bone marrow harvest.

Example 7 Formulation of the Chemotactic Hematopoietic Stem Cell ProductComprising CD34+ Cells

The chemotactic hematopoietic stem cell product is formulated in 10-mlof saline (0.9% Sodium Chloride, Injection, USP, Hospira, Cat# 7983-09)supplemented with 1% HSA (Human Albumin USP, Alpha, Cat. # 521303)(“Infusion Solution”) and at least 10% autologous serum. In addition,there may be some trace amount of materials (quantities not determined)in the Chemotactic hematopoietic stem cell product that are used andleft over during the product processing. These materials include:Dulbecco's Phosphate Buffered Saline-Ca⁺⁺, Mg++ Free (D-PBS) (Baxter,Cat. # EDR9865), Sodium Citrate (Baxter/Fenwal, Cat. # 4B7867),Hetastarch (Abbott Laboratories, Cat. # 0074-7248-03), IVIg (Gammagard®Immune Globulin Intravenous, Baxter, Cat. # 060384) and the reagents inthe Isolex® 300i Stem Cell Reagent Kit (Baxter, Cat. # 4R9734) includinganti-CD34 monoclonal antibody, stem cell releasing agent and Sheepanti-mouse magnetic beads.

Example 8 Transporting Chemotactic Hematopoietic Stem Cell Product tothe Catheterization Facility

The chemotactic hematopoietic stem cell product that meets the releasecriteria will be loaded into a sterile 10 cc syringe in a Class 100biological safety cabinet located within a controlled asepticenvironment, e.g., at minimum, a Class 100,000 cell processing facility;class 10,000 is preferable, but not required. The chemotactichematopoietic stem cell product will be suspended in 10-ml PBSsupplemented with HSA and the container labeled in accordance withrelease criteria. There are to be four dosing cohorts consisting of fivesubjects each in each cohort. The first will receive about 5×10⁶ CD34⁺cells, the second about 10×10⁶ CD34⁺ cells, the third about 20×10⁶ CD34⁺cells and the fourth about 30×10⁶ CD34⁺ cells. Subjects in cohorts 2-4with inadequate CD34⁺ cell quantities to meet the assigned cohort dosewill be added to a prior cohort at the greatest possible CD34⁺ celldose. The loaded infusion syringe will be attached to a four-waystopcock along with a flushing syringe, capped and have safety guardsapplied to prevent leakage. The delivery apparatus will be sealed in adouble sterile bag and placed in a secure transportation box fortransportation to the cardiac catheterization facility. Followingrelease of the chemotactic hematopoietic stem cell product and cohortassignment, the chemotactic hematopoietic stem cell product will beshipped to the catheterization site for direct infarct-related arteryinfusion (“intravascular administration”).

Example 9 Intra-Coronary Infusion of Chemotactic Hematopoietic Stem CellProduct

Upon notification from the cell processing facility that the chemotactichematopoietic stem cell product has been released for infusion (seesupra), the subject/patient will be scheduled to arrive at thecatheterization facility at a time to coincide with the arrival of thechemotactic hematopoietic stem cell product.

Cardiac enzymes (brain natriuretic peptide (BNP), troponin and CPK MB),complete blood counts, a full chemistry panel (renal and liver functiontest) and an EKG will be performed just prior to chemotactichematopoietic stem cell product infusion. Clinical assessment of thestage of heart failure according to the New York Heart Association's(NYHA) functional classification system will be recorded.

Upon receipt of the chemotactic hematopoietic stem cell product andfinal quality assurance release (by facsimile) for infusion, the subjectwill undergo cardiac catheterization as detailed above. Coronaryarteriography will be performed to assess for patency (meaning openness,freedom from blockage) of the infarct related artery and Thrombolysis inMyocardial Infarction (TIMI) angiographic flow. A balloon catheter overa wire will be placed in the stented segment of the infarct relatedartery. Any appropriate balloon dilatation catheter having an internaldiameter of at least about 0.36 mm compatible with the chemotactichematopoietic stem cell product infusion can be used. After positioning,the balloon wire will be removed. The chemotactic hematopoietic stemcell product delivery apparatus will be removed from the transportationcase.

The delivery apparatus will be in a sterile bag and have safety blocksattached to the infusion syringe (containing the chemotactichematopoietic stem cell product) and the flushing syringe. The apparatusconsists of the infusion syringe (containing 10 ml of the chemotactichematopoietic stem cell product) and the flushing syringe (containing 6ml of flushing solution) wherein both are attached to a sterile four-waystopcock. The entire delivery apparatus should be shaken gently toresuspend the CD34⁺ cells in the infusion solution. The flushing syringeis used to eliminate all air bubbles in the apparatus (to prevent airemboli) and the delivery apparatus then attached to the balloondilatation catheter via the stopcock.

Delivery of the chemotactic hematopoietic stem cell product to thesubject by infusion will proceed as follows. First, with the stopcockopen between the flushing syringe (6 ml solution) and the central lumenof the balloon catheter, 1 ml of flushing solution should be infused(after removal of the guard) into the central lumen of the catheter over15 seconds. Second, the balloon should be inflated at two atmospheres ofpressure within the stent to avoid damage to the coronary arteryendothelium and then the stopcock valve adjusted to allow infusion ofthe chemotactic hematopoietic stem cell product distal to the inflatedballoon (after removal of the guard). With the balloon inflated, about 3cc to about 4 cc from the infusion syringe will be infused by hand overa period of about 30 seconds to about 45 seconds (to be timed anddocumented). The balloon will remain inflated to allow adhesion of theCD34⁺ cells and to prevent back flow for a total of about 2 minutes toabout 3 minutes (including the time for infusion). In between infusions,the balloon will remain deflated for 3 minutes to allow restoration ofblood flow (reperfusion). It is expected that 3 infusions will berequired to empty the infusion syringe. Third, upon completion ofinfusing the chemotactic hematopoietic stem cell product and with theballoon deflated, the valve on the stopcock will be adjusted to allowfilling of the infusion syringe from the flushing syringe. Finally, withthe balloon inflated (about 2 minutes to about 3 minutes), the 4 ml offlushing solution now in the infusion syringe will be infused over aperiod of about 30 seconds to about 45 seconds to dislodge any residualCD34⁺ cells from the syringe and catheter into the IRA circulation. Thecatheter then is removed.

An infusion related ischemia (inadequate blood flow) assessment will beperformed during the first 24 hours after chemotactic hematopoietic stemcell product infusion. An EKG at about 12 hours and at about 24 hoursand analytical chemistry of cardiac enzymes (BNP, troponin and CPK MB)about every 8 hours for about 24 hours will be obtained. Arrhythmiaassessment (24 hour Holter monitor) will be performed immediatelypost-chemotactic hematopoietic stem cell product infusion. Routinetransthoracic echocardiography to evaluate global and regional leftventricular function will be performed prior to the subjects dischargeafter chemotactic hematopoietic stem cell product infusion.

All subjects will be provided with digital thermometers and a log bookto record twice daily temperatures for 30 days post infusion of thechemotactic hematopoietic stem cell product. Subjects will be instructedto notify the investigator site immediately for temperatures recordedabove 100.5° F. Rapid follow-up with appropriate cultures andradiographic assessments will be performed according to routine clinicalstandards. Documented bacterial infections will be reported to the IRBand the FDA.

Additional follow-up visits for safety assessments will include visitsat 1 week and 2 weeks after product administration. Visit assessmentswill include a comprehensive medical history and physical examination,EKG, complete blood counts, full chemistry panel (renal and liverfunction test), and measure of serum cardiac markers (BNP, troponin andCPK MB). Clinical assessment of NYHA functional class will be recordedon week 1 and 2.

At 1 week post infusion, routine transthoracic echocardiography is to beperformed.

At 4 weeks post chemotactic hematopoietic stem cell product infusion, anEKG and cardiac enzymes (BNP, troponin and CPK MB) will be obtained.Routine transthoracic echocardiography to evaluate global and regionalleft ventricular function also is to be performed. A 24 Holter monitorwill be used to assess for arrhythmias. Clinical assessment of NYHAfunctional class will be recorded. Treadmill exercise testing using asymptom limiting Bruce protocol will be performed as well.

At about 3 months and about 6 months post chemotactic hematopoietic stemcell product infusion, a 24 hour Halter monitor will be performed.Clinical assessment of NYHA functional class will be recorded. At about6 months post chemotactic hematopoietic stem cell product infusion, asymptom limited treadmill exercise testing using the Bruce protocol willbe recorded.

A safety assessment at about 12 months post chemotactic hematopoieticstem cell product infusion will include a comprehensive medical historyand physical examination, EKG, complete blood counts, full chemistrypanel (renal and liver function test), and measure of serum cardiacmarkers (BNP, troponin and CPK MB). Routine transthoracicechocardiography to evaluate global and regional left ventricularfunction also is to be performed. A 24 hour Holter monitor will beperformed. Clinical assessment of NYHA functional class will berecorded.

Statistical Analysis

A paired design, where each subject serves as his or her own control,will be used in some embodiments. Differences between before and aftertreatment, per subject, will be analyzed for each of the four numericcardiac functions (i.e., myocardial contractility; end systolic volume,end diastolic volume; and perfusion). Linear regression analysis will beused to assess the significance of increased dosing levels. The nullhypothesis is that the slope of the regression line (dosing levelserving as the independent variable and the “after” minus the “before”difference serving as the dependant variable) is equal to zero. Thepower of rejecting a false null hypothesis is 0.68 at the 0.05 alphalevel of significance for a high correlation of 0.5 between dosing andimprovement in cardiac function. The 95% confidence interval about theslope of the regression line will be used to assess the medicalsignificance of the increase in dosing level. If the slope of theregression line is not significantly different from zero but theintercept of the regression line is different from zero, then alltreatment groups will be combined and a paired t-test will be performedto assess the overall treatment effectiveness. The null hypothesis isthat the mean of the differences is equal to zero. A Wilcoxonsigned-ranks test also will be performed as an additional test todetermine the treatment effectiveness. This test is more powerful(rejecting a false null hypothesis) than a t-test if the observationsare not normally distributed. The power of the t-test is 0.79 forrejecting a false null hypothesis at the alpha level of 0.05 and thetreatment having a medium size effect (an effect large enough to bediscernable by the naked eye). The medical significance of the treatmenteffect size will be determined by computing a 95% confidence intervalabout the mean of the differences (the true mean of the differences willlay in this interval in 95% of tested samples).

To assess improvement in perfusion, logistic regression will be usedwith dosing level as the independent variable and perfusion change(1=yes, 0=no) as the dependant variable. Odds ratios of the four dosinglevels will be computed separately with 5.0×10⁶ cells serving as theindex group.

A binomial test will be used to assess the significance of CD34⁺ celldosing on perfusion. It is expected that there will be no spontaneousimprovement in a perfusion defect if present on the baseline perfusionscan. Therefore, any clinically significant improvement in a perfusiondefect when assessed at 6 months and compared to baseline will beconsidered a treatment effect.

A concurrent group (non-treated controls) meeting eligibility but notreceiving CD34⁺ cells will be evaluated similar to the treated group andassessed for significant improvement in cardiac function/perfusion. Eachstudy site will alternate accrual of treated and non-treated controls. Acoin flip will be used to determine the initial (treated or non-treated)subject sequence at each site. Comparison of outcomes between treatedand non-treated groups will be made. The core lab will be blindedregarding treatment or no-treatment.

An assessment will be performed to determine if a correlation existsbetween clinical outcome and cell content (CD34⁺) and/or in vitro colonygrowth (CFU-GM, CFU-GEMM, BFU-E), CXCR-4 mobility, and CXCR-4 and/orVEGF surface antigen expression.

A total of 20 subjects will receive the chemotactic hematopoietic cellproduct of the present invention. There will be four dose cohorts (about5×10⁶, about 10×10⁶, about 20×10⁶, and about 30×10⁶ CD34⁺ cells). If thechemotactic hematopoietic stem cell product content in any subject isnot sufficient for the assigned cohort, that subject will be reassignedto a prior cohort at the greatest possible dose. Subjects having fewerthan 5×10⁶ CD34⁺ cells available for infusion will be removed fromstudy, will not undergo repeat catheterization and will not be countedas part of the 20-subject study group. In addition, if the chemotactichematopoietic cell product of the present invention does not meetrelease criteria, the subject will not receive the cell product and willnot be counted as a study candidate to be replaced by the next subject.In any cohort dosing group, if a subject experiences an acute (meaningimmediate to about 7 days post infusion) unexpected toxicity consideredto (probably) be a result of the cell product infusion, dose escalationwill be halted and 3 additional subjects will be accrued to that doselevel. If no other unexpected toxicity is observed, then dose escalationwill resume, however the total of 20 subjects will not be exceeded. Ifanother toxicity occurs at that dose level, then all subsequent subjectswill be accrued to the next lower dose level.

The chemotactic hematopoietic stem cell product of the present inventionwill not be administered to any subject in the higher dose cohort untilall the subjects from the prior dose cohort have completed theirfollow-up assessments two weeks after product administration.

Example 10 Experimental Results of Preliminary Studies

A series of preliminary preclinical studies have been performed in anattempt to accomplish the following goals:

(1) Optimize the manufacturing process for the Mini bone-Marrow Harvest(MMH);

(2) Evaluate the stability of the inbound MMH product and the outboundhematopoietic cell product.

(3) Evaluate the internal diameter allowance and safety of thecatheters;

(4) Evaluate the compatibility of the cell product with the cathetersintended to be used in the study; and

(5) Evaluate the suitability of using the supernatant of the finalhematopoietic cell product to represent the final hematopoietic cellproduct for stability testing.

Study 1: Optimizing the Manufacturing Process for the Mini Bone-MarrowHarvest (MMH);

The effect of key manufacturing variables on the yield of viable CD34cells from representative bone marrow products was evaluated. A total ofsix (6) volunteer donors over the age of 45 (based on a range of 45-57)and three under 30 years of age (based a range of 21-28) agreed todonate an average of 45 ml (based on a range of 31 ml-54 ml) bone marrowand provided written Informed Consent for the procedure. The marrowaspiration technique employed was identical to that to be performed forthe clinical scale MMH (see Example 3, supra). As shown in Table 2, thecell counts of nucleated cell (NC) and CD34+ cells of Mini bone-MarrowHarvest (“MMH”) derived cells collected from volunteer donors appearedto be age related.

TABLE 2 Effect of donor age on nucleated cell yield of the MMH. Donorage group Over 45 (45-57) Under 30 (23-28) Volume of Viability CD34cells Volume of MMH Viability CD34 cells Donor MMH (ml) (%) (10⁵ per ml)(ml) (%) (10⁵ per ml) 1 31.30 83.85 1.27 48.00 96.90 7.98 2 43.50 97.423.89 50.60 96.28 11.60  3 51.50 85.74 1.37 39.90 87.17 5.99 4 47.5080.95 1.76 — — — 5 53.70 98.21 5.58 — — — 6 44.90 96.36 4.48 — — — Avg.45.40 90.42 3.06 46.17 93.45 8.52

The average cell count of the bone marrow products from older donors(N=6) was 28.4×10⁶ (based on a range of 15.8×10⁶-49.5×10⁶) nucleatedcells per ml [“NC/ml”], with an average viability, as determined by7-AAD dye exclusion and flow cytometry, of 90.42% (based on a range of80.95%-98.21%) and CD34+ content of 3.06×10⁵/ml (based on a range of1.27×10⁵/ml -5.58×10⁵/ml). In the younger subject group (N=3), theaverage cell count collected from marrow aspiration was 46.2×10⁶ NC/ml(based on a range of 39.9×10⁶ NC/ml-50.6×10⁶ NC/ml), with an average7-AAD viability of 93.5% (based on a range of 87.17%-96.90%) and totalCD34⁺ content of 8.5×10⁵/ml (based on a range of 5.99×10⁵ CD34+cells/ml-11.60×10⁵ CD34+ cells/ml).

Red Cell Depletion and CD34 Selection

TABLE 3 CD34+ cell recovery after RBC depletion of MMH from older agegroup (4557) donors. Donor 1 2 3 4 5 Average Method of RBC HetastarchBuffy Buffy Buffy Buffy — depletion coat coat coat coat CD34+ cell % in1.09 1.64 1.63 1.45 1.99 1.58 MMH: Pre-RBC depletion CD34+ cell % in1.33 1.55 1.51 1.61 1.84 1.57 MMH: Post-RBC depletion CD34+ cellrecovery 65.68 92.36 80.66 78.79 81.67 79.83 post RBC depletion (%)

As shown in Table 3, following red cell depletion of the MMH-derivedbone marrow products collected from the older donors, an average of79.83% (based on a range of 65.68%-92.36%) of the CD34 cells from theinitial MMH was recovered. There was no significant difference betweenthe initial CD34 cell purity (1.58%, based on a range of 1.09%-1.99%)and that following red cell depletion (1.57%, based on a range of1.33%-1.84%).

TABLE 4 CD34+ cell recovery, purity, CXCR-4 migratory activity,viability and hematopoietic CFU growth immediately after Isolexprocessing of MMH from older age group (age 45-age 57) donors. Donor 1 23 4 5 Average Storage time (hours) at 0 0 0 12 10.50 — 4° C.-8° C. CD34+cell recovery 32.36 29.09 15.31 43.60 40.20 32.11 (%) CD34+ cell purity(%} 76.76 73.64 71.66 72.52 72.01 73.32 CD34+ cell viability 98.49 93.8097.38 98.28 98.39 97.27 CD34+ cell CXCR-4 22.10 2.60 22.00 19.90 19.7017.26 migratory activity (%) Hematopoietic CFU/100 27.5 25.0 18.9 17.021.00 21.9 CF34+ cells cultured

As shown in Table 4, following CD34 selection using the Isolex system,which includes immunomagnetic Dynabeads® and anti-CD34 MAb, we recoveredan average of 32.11% (based on a range of 15.31%-43.60%) of the CD34cells with an average purity of 73.32% (based on a range of71.66%-73.64%) and an average viability of 97.27% (based on a range of93.80%-98.49%). In addition, these CD34+ cells displayed an average of17.26% (based on a range of 2.60%-22.10%) CXCR-4 migratory abilityimmediately after selection and were capable of generating hematopoieticcolonies (21.89 colonies/100 CD34+ cells plated (based on a range of17.0 colonies/100 CD34+ cells plated-27.5 colonies/100 CD34+ cellsplated) in MethoCult culture.

Study 2: Evaluation of the Stability of the Inbound Mini-Bone MarrowHarvest and of the Outbound Chemotactic Hematopoietic Cell Product:

A series of experiments, using healthy volunteers, was performed inorder to evaluate the stability of the inbound MMH and of the outboundchemotactic hematopoietic stem cell product of the present invention.Assessment of the functional viability of the inbound and outboundproducts was evaluated by cell viability (7-AAD), SDF-1/CXCR-4 mediatedCD34+ cell migration, and the ability to form hematopoietic colonies inmethylcellulose (CFU colony forming ability).

To evaluate the inbound product stability for shipping and logisticpurposes and for coordination with clinical schedules, MMH products werestored at 4° C. to 8° C. as indicated. To evaluate the outbound productstability for shipping and logistic purposes, the chemotactichematopoietic stem cell product comprising isolated CD34+ cells enrichedfollowing MMH was stored at 4° C. to 8° C. as indicated.

In preliminary studies, cells either were processed immediately ormaintained at 4-8° C. for 12 hours prior to processing to evaluate theimpact of shipping and logistic duration on the manufacture a suitablecell product for infusion. Despite the duration of storage prior toprocessing (inbound product expiration), the results did not varysignificantly (data not shown).

In another series of experiments, cells were stored at about 4° C. toabout 8° C. for 12 hours and about 24 hours prior to reassessment tosimulate products infused at about 36 hours and at about 48 hours,respectively, following MMH.

TABLE 5 CD34+ cell viability, growth and CXCR-4 migratory activity13-13.5 hours after Isolex processing of MMH. Donor 1 2 Average CD34+cell viability (%) 97.59 96.90 97.24 CD34+ cell CXCR-4 migratoryactivity (%) 7.70 7.50 7.60 Hematopoietic CFU/100 CD34+ cells cultured18.00 25.00 21.5

As shown in Table 5, the isolated CD+34 cells of the chemotactichematopoietic stem cell product had an average viability of 97.24%(based on a range of 96.90%-97.59%) and average CXCR-4-mediatedmigratory capacity of 7.60% (based on a range of 7.50%-7.70%). As shownin Table 6, after storage for an average of 26.3 hours (based on a rangeof 26.0 h-26.5 h), these cells had an average viability of 96.81% (basedon a range of 96.39%-97.22%) and an average CXCR-4-mediated migratorycapacity of 4.75% (based on a range of 4.50%-5.00%). Further, the cellsstill maintained their ability to generate hematopoietic colonies invitro.

TABLE 6 CD34+ cell viability, growth and CXCR-4 migratory activity26.0-26.5 hours after Isolex processing of MMH. Donor 1 2 Average CD34+cell viability (%) 97.22 96.39 96.81 CD34+ cell CXCR-4 migratoryactivity (%) 4.50 5.00 4.75 Hematopoietic CFU/100 CD34+ cells cultured28.00 14.00 21.00

Thus, an average of 13.3 hours (based on a range of 13.0 h-13.5 h) afterCD34+ cell selection, representing 26.0-26.5 hr post-MMH, the CD34+ cellpopulation had an average viability of 97.24% (based on a range of96.90%-97.59%), with average CXCR-4 mediated migratory capacity of 7.60%(based on a range of 7.50%-7.70%). At an average of 26.3 hours (based ona range of 26.0 h-26.5 h) following MMH, the average viability of thecells was 96.81% (based on a range of 96.39%-97.2%) and maintained anaverage CXCR-4-mediated migratory capacity of 4.75% (based on a range of4.50%-5.00%).

Formulation of the composition of the present invention comprising thisproduct occurred an average of 8 hours (8.63±1.80 N=4) hours after MMHcollection, and infusion occurred within 24 hours of MMH.

TABLE 7 CD34+ cell viability as a function of time after MMH: 12-hourin-dating and 48 hour outdating (all time points measured fromcompletion of MMH.) CD34+ cell viability (%) Time (h) after MMH Average(SD) A B C D (SD) 98.22 97.13 97.60 99.00 97.99 (0.29) 24 95.32 97.76 —— 96.54 (1.73) 33 91.92 96.32 95.90 80.00 91.04 (7.62)

In a subsequent experiment, four (4) MMH products (A-D) were collectedand stored at 4° C. for an average of 12.8 hours (based on a range of12.5 h-13.0 h) before the CD34+ cells were isolated by the Isolexprocedure. This group, representing the “12 hour in-date” group (meaningthat the product was formulated within the in-date time of about 12hours), was evaluated for functional viability out-date at “24 hours”(22.9 h±1.63, N=4), “33 hours” (33.38±1.11, N=2), and “48 hours”(48.33±0.82, N=4) post MMH harvest. The data, summarized in Tables 7-9,demonstrate that following MMH, the chemotactic hematopoietic stem cellproduct comprising enriched CD34+ cells maintains 1) high viability(>90.0% average viability, Table 7), 2) 76.85% (±21.66) of theirSDF-1VEGF/CXCR-4 mediated migratory ability (Table 8), and 3) theirability to form hematopoietic colonies in vitro (Table 9), respectively.

Table 8 shows SDF-1/VEGF/CXCR-4 mediated CD34+ cell migration (%migrating CD34+ cells as a function of time after MMH: 12-hour in-datingand 48-hour outdating (all time points measured from completion of MMH).For the purposes of determining the impact of time post-MMH on themigratory ability of the CD34+ cells, time point “X” was considered thereference point, as this was determined to represent the earliest timepoint following MMH at which cells reasonably could be expected to bereturned to the subject in a finished formulation. The remainingmigratory activity at the following time points (Y=33 hours, Z=48 hours)was calculated as percent migratory ability remaining following the 24hour (X) time point.

TABLE 8 SDF-1/VEGF/CXCR-4 mediated CD34+ cell migration (% migratingCD34+ cells as a function of time after MMH: 12-hour in-dating and48-hour outdating (all time points measured from completion of MMH).Migrating CD34+ cells (%) Average Time (h) after MMH A B C D (SD) 24 (X)20.00 18.50 21.50 36.00 24    (8.09) % Remaining 100.00 100.00 100.00100.00 100.00  (0)   33 (Y) 21.80 10.50 — — 16.15  (7.99) *% Remaining109.00 56.76 — — 82.88 (36.94) 48 (Z) 8.80 17.00 17.50 31.00 18.58 (9.19) ^(@)% Remaining 44.00 91.89 81.40 86.00 75.85 (21.66) *= (Y ÷ X)× 100% ^(@)= (Z ÷ X) × 100%

Table 9 shows the number of colony forming units (CFU) per 100 viableCD34+ cells plated as a function of time after MMH: 12-our in-dating and48 hour-out-dating (all time points measured from completion of MMH.

TABLE 9 # of CFU per 100 viable CD34+ cells plated Time (h) Averageafter MMH A B C D (SD) 24 13.00 30.00 37.00 39.00 29.75 (11.81) 33 12.0034.00 — — 23.00 (15.56) 48 15.00 30.00 20.00 8.00 28.25 (14.57)

In an attempt to extend both the in-date and out-date stabilityparameters for the chemotactic hematopoietic stem cell product of thepresent invention comprising CD34+ cells from 12-hours (in-date) andfrom 48-hours (out-date) (12/48), respectively, to 24-hours (in-date)and 72-hours (outdate) (24/72), respectively, CD34 cells were purifiedabout 12 hours after MMH harvest (12 hour in-date) and about 24 hoursafter MMH harvest (24 hour in-date) and analyzed for functionalviability at about 48 hours and at about 72 hours total time from MMH totime of testing/anticipated infusion (48 hour out-date and 72 hourout-date, respectively). Specifically, the functional viabilitycharacteristics of two MMH/chemotactic hematopoietic stem cell productsof the present invention were evaluated at 48 hours and 72 hours. Theresulting data were further compared to the same indices derived at theprevious 12/48 time points (Tables 7-9).

Tables 10-12 show that at 33 hours (based on 32.5±0.71, N=2), 48 hours(based on one data point at 49 hours), and at 72 hours (based on 72.5h±0.71, N=2), the isolated CD34+ cells of the chemotactic hematopoieticstem cell product of the present invention maintain 1) over 90%viability (Table 10), 2) 102.19±32.69% of their SDF-11VEGF/CXCR-4mediated migratory ability (Table 11), and 3) their ability to generatehematopoietic colonies in vitro (Table 12).

TABLE 10 CD34+ cell viability as a function of time after MMH: 24-hin-dating and 72-h outdating (all time points measured from completionof MMH) CD34+ cell viability (%) Time (h) after Average MMH A B (SD) 3398.00 99.00 98.50 (0.71) 48 — 97.00 97.00 (—) 72 91.00 97.00 94.00(4.24)

TABLE 11 SDF-1/VEGF/CXCR-4 mediated CD34+ cell migration (% populationof migrated CD34+ cells as a function of time after MMH): 24-h in-datingand 72-h outdating (all time points measured from completion of MMH):Time (h) after Migrating CD34+ cells (%) MMH Average (SD) A B (range) 338.20 14.05 11.13 (2.93) % Remaining 100.00 100.00 100.00 (0.00) 48 —18.61 18.61 (—) % Remaining — 132.46 132.46 (—) 72 5.70 18.95 12.33(6.63) % Remaining 69.51 134.88 102.19 (32.69)

The % remaining ratios in Table 11 were determined as in table 8 above.

TABLE 12 Number of CFU per 100 viable CD34+ cells plated as a functionof time after MMH: 24-h in-dating and 72-h outdating (all time pointsmeasured from completion of MMH) Time (h) after # of CFU per 100 viableCD34+ cellsplated MMH Average (SD) A B (range) 33 26.00 28.50 22.25 (1.25) 48 — 16.80 16.80 (—) 72 14.50 27.50 21.00 (6.5)

Further evaluation of the functional viability parameters of thechemotactic hematopoietic stem cell product comprising isolated CD34+cells of the present invention (“clinical product”) at 8 hours (8.6h±1.80, N=4), 12 hours (12.87 h±1.92, N=4), 32 hours (one time point at33.5 h), 48 hours (47.50 h±2.5, N=2), and 72 hours (71.5 h±0.50, N=2)after MMH shows that after 72 hours, the product retains its 1)viability (Table 13), 2) SDF-mediated migratory ability (Table 14) and3) ability to form hematopoietic colonies in vitro (Table 15),equivalent to the 24-hour time point.

TABLE 13 Clinical Product Experience: CD34+ cell viability as a functionof time after MMH. CD34+ cell viability (%) Time (h) after Average MMH AB C D (SD) 8 98.30 99.08 90.00 96.45 95.96 (4.12) 12 98.89 96.96 99.0099.43 98.57 (1.10) 33 — 93.42 — — 93.42 48 — 93.15 91.58 — 92.37 (1.11)72 — 91.25 89.25 — 90.30 (1.48)

TABLE 14 Clinical Product Experience: SDF-1/VEGF/CXCR-4 mediated CD34+cell migration (% migrating CD34+ cells as a function of time after MMH)Migrating CD34+ cells (%) Average Time (h) after MMH A B C D (SD) 12 (X)14.31 13.08 9.74 31.73 17.97 (11.34) % Remaining 100.0 100.0 100.0 100.0100.0  (0)   33 (Y) — 6.17 — —  6.17 *% Remaining — 47.17 — — 47.17 48(Y) — 4.88 8.21 —  6.55  (2.35) *% Remaining — 37.30 84.29 — 60.79(23.49) 72 (Y) — 3.7 6.6 —  5.15  (2.05) *% Remaining — 28.29 21.19 —24.74  (3.55) *= (Y ÷ X) × 100%

All remaining ratios were calculated as in Table 8 above.

TABLE 15 Clinical Product Experience: # of CPU per 100 viable CD34+cells plated as a function of time after MMH # of CFU per 100 viableCD34+ cells plated Time (h) Average after MMH A B C D (SD)  12. 98.1433.30 24.00 22.50 44.49 (36.09) 33 — 16.50 — — 16.5  48 — 19.56 20.50 —20.03  (0.66) 72 — 20.45 21.19 — 20.82  (1.10)

Based on these data, extension of the in-dating to 24 hours (from12-hours) and the out-dating to 72 hours (from 48 hours) for the CD34+cell clinical product of the present invention is justified.

FIG. 1 indicates the equivalence of the functional viability of thechemotactic hematopoietic cell product of the present invention at 72hours to the same indices evaluated at 48 hours.

Study 3: Catheter Safety.

The viability and potential efficacy of the chemotactic hematopoieticstem cell product of the present invention comprising potent CD34+ cellsdepends on the cells maintaining their potency as they pass through acatheter. The catheter used in the methods of the present invention hasan internal diameter of at least 0.36 mm. Any type of catheter having aninternal diameter of at least 0.36 mm may be effective in delivering thepharmaceutical compositions of the present invention.

In one embodiment, the catheter is a balloon catheter. Balloon cathetersafety studies were conducted to determine whether high cellconcentrations and repeated perfusions adversely affect cell viability,cell recovery or catheter integrity. Non-mobilized peripheral bloodprogenitors were used in order to obtain an adequate number of cells toperform the analysis. Catheters were assessed for infusion of the cellproduct of the present invention comprising selected CD34+ cells throughthe IRA. None of the 0.36 mm internal diameter catheters testedadversely affected CD34+ selected cell viability, growth in culture, ormobility in CXCR-4 assays.

TABLE 16 Viability of CD34+ cells before and after infusions through thecatheters. Viability (%) Catheter Condition 1 2 3 4 5 — Pre-infusion81.45 Raptor After 1st infusion 84.29 70.94 87.89 88.02 84.68 After 2ndinfusion 83.00 87.44 86.39 79.91 83.18 Sprinter After 1st infusion 93.3991.09 84.13 88.28 81.68 After 2nd infusion 91.89 91.08 84.88 77.65 77.73Voyager After 1st infusion 94.21 86.21 83.08 77.53 69.68 After 2ndinfusion 88.03 84.71 79.27 78.11 76.80 Maverick After 1st infusion 90.0089.76 90.79 85.49 81.31 After 2nd infusion 90.94 87.38 81.98 80.09 85.47

As shown in Table 16, in all catheters tested, average CD34⁺ cellviability was at or above 70% following passage through the catheters.

To demonstrate that infusion of the CD34+ cell product does not pose anysafety breach of the catheter used and that a significant percentage ofcell product does not adhere to the interior walls of the catheter,catheters were challenged with repeat infusions of a CD34+ cell producthaving a considerably higher cell concentration than that usedclinically. Four brands of catheters (Sprinter, Voyager, Maverick andRaptor) were evaluated using 5 catheters of each type. Non-mobilizedapheresis products were used in order to obtain an adequate number ofcells to perform the analysis. A cell concentration greater than threetimes that planned as treatment doses for the trial, i.e., 160×10⁶nucleated cells containing CD34+ cells in 10 ml of infusion solution,was passed twice through each catheter. The average CD34+ cell recoverywas 100.59% (based on a range of 76.99% to 228.70%) following passagethrough the catheters.

All twenty catheters were tested for integrity using a methylene bluedye leak test after two perfusions with the nucleated cells. There wasno evidence of leakage and the contact points and catheter tips werenormal upon inspection.

As shown in Table 17a and 17b, the effect on the cells of theirperfusion through a catheter appears to be independent of catheter modeland make among those catheters tested and was independent of the amountof time the cells were stored either prior to processing and/or afterCD34+ cell selection and prior to perfusion, resulting in a finalformulation containing an average recovery of 96.0% (range 80.8%-102.2%)of the CD34+ cells (Table 17b) and 86.36% of the CD45+ cells perfusedthrough the catheter. Further, the average viability of the cells was96.5% (range 92.5%-98.6%, N=16); the cells maintained both CXCR-4migratory capacity (data not shown) and their ability to formhematopoietic colonies in methylcellulose (average 25.8 CFU/100 cellsseeded (range 21.0%-30.5%)

TABLE 17a CD45 cell recovery and viability after being infused throughthe catheters. 1 2 3 4 5 Average R'd Re- R'd R'd R'd R'd R'd CatheterCondition Recovery viab covery viab Recovery viab Recovery viab Recoveryviab Recovery viab Raptor After 1^(st) 69.68% −1.35% 78.67% 2.08% 72.14%−4.55% 80.54% 1.83% 73.21% −2.13% 74.85% −0.82% infusion (30.83%)(2.53%) After 2^(nd) 97.91% −8.55% 81.84% −4.76% 142.98% 3.28% 107.82%−8.48% 94.08% 0.08% 104.93% −3.69% infusion (47.60%) (4.94%) SprinterAfter 1^(st) 76.74% −0.60% 68.56% 4.01% 72.63% 5.29% 73.61% 6.06% 66.83%8.31% 71.67% 4.61% infusion (29.48%) (3.51%) After 2^(nd) 78.82% 2.86%85.40% 0.98% 90.29% −1.02% 82.22% 6.50% 91.61% 0.00% 85.67% 1.86%infusion (35.30%) (2.76%) Voyager After 1^(st) 87.38% 1.58% 83.93%−0.36% 103.58% 0.93% 95.82% 4.52% 131.55% −4.39% 100.45 0.46% infusion(44.39%) (2.91%) After 2^(nd) 82.70% 7.01% 69.34% 15.90% 69.54% 10.40%89.04% 0.27% 69.03% 7.50% 75.93% 8.22% infusion (32.11%) (6.09%)Maverick After 1^(st) 73.97% 1.58% 87.01% 0.42% 78.31% 0.69% 75.53%2.61% 77.22% 2.95% 78.41% 1.65% infusion (32.33%) (1.21%) After 2^(ndX)152.35% −5.06% 73.44% 2.78% 80.85% −3.92% 97.10% −2.97% 91.11% −2.07%98.97% −2.25% infusion (49.11%) (2.85%) Average of all 86.36% 1.26%catheters: ^(a)Recovery of CD45+ cells = (# of CD45 cells afterinfusion + # of CD45 before infusion) × 100% ^(b)Reduction of CD45+ cellviability = [1 − (CD45+ cell viability % after infusion ÷ CD45+ cellviability % before infusion)] × 100%

TABLE 17b CD34 cell recovery and viability after being infused throughthe catheters. Catheter 1 2 3 used Condition Recovery^(a) R'd viab^(b)Recovery R'd viab Recovery R'd viab Raptor After 1^(st) infusion 116.49%−3.48% 121.62% 12.91% 110.89% −7.91% After 2^(nd) infusion 91.66% 1.53%85.18% −23.26% 122.47% 1.71% Sprinter After 1^(st) infusion 89.19%−14.66% 83.34% −11.83% 102.72% −3.29% After 2^(nd) infusion 103.52%1.61% 99.82% 0.01% 82.11% −0.89% Voyager After 1^(st) infusion 81.02%−15.67% 96.08% −5.84% 90.16% −2.00% After 2^(nd) infusion 106.48% 6.56%81.66% 1.74% 95.04% 4.58% Maverick After 1^(st) infusion 76.99% −10.50%101.79% −10.21% 98.62% −11.46% After 2^(nd) infusion 228.70% −1.05%88.66% 2.65% 103.35% 9.70% Average Catheter 4 5 Recovery R'd viab usedCondition Recovery R'd viab Recovery R'd viab (SD) (SD) Raptor After1^(st) infusion 97.55% −8.06% 96.14% −3.97% 108.54% −2.10% (45.46%)(7.79%) After 2^(nd) infusion 111.33% 9.21% 98.96% 1.78% 101.92% −1.81%(43.73%) (11.14%) Sprinter After 1^(st) infusion 84.57% −8.39% 88.65%−0.28% 89.69% −7.69% (37.26%) (6.16%) After 2^(nd) infusion 114.87%12.05% 100.45% 4.84% 100.15% 3.52% (42.22%) (4.90%) Voyager After 1^(st)infusion 82.73% 4.82% 89.32% 14.46% 87.86% −0.85% (36.28%) (10.13%)After 2^(nd) infusion 94.81% −0.75% 91.01% −10.23% 93.80% 0.38% (39.12%)(5.86%) Maverick After 1^(st) infusion 112.58% −4.96% 96.05% 0.18%97.21% −7.39% (41.34%) (5.34%) After 2^(nd) infusion 89.35% 6.31%117.63% −5.12% 125.54% 2.50% (73.48%) (5.33%) Average of all 100.59%−1.68% catheters: ^(a)Recovery of CD34+ cells = (# of CD34 cells afterinfusion + # of CD34 before infusion) × 100% ^(b)Reduction of CD34+ cellviability = [1 − (CD34+ cell viability % after infusion ÷ CD34+ cellviability % before infusion)] × 100%

Collectively these experiments demonstrate that the serial passage of achemotactic hematopoietic stem cell product comprising CD34+ cellsthrough a cardiac catheter with an internal diameter of at least about0.36 mm does not adversely affect either catheter integrity or CD34+cell potency, i.e., CD34+ cell viability, CFU colony growth, or CD34+CXCR+ mediated migratory capacity/mobility.

Study 4: Compatibility of the Cell Product with the Catheters

To further test the compatibility of the chemotactic hematopoietic stemcell product comprising CD34⁺ cells with each of the catheters that maybe used for delivery of the cell product in the study, cell productswere tested after multiple passages through each catheter type toevaluate the effects of extreme conditions of stress that would begreater than those expected during the treatment protocol.

At 48 hours post-MMH harvest, the chemotactic hematopoietic stem cellproduct comprising a range of about 5.73×10⁶ CD34+ cells to about21.10×10⁶ CD34+ cells (i.e., dosages reflective of the treatment cohort)obtained from individual donors was infused sequentially through threecatheters of the same brand, one type of catheter for each donor(Sprinter, Voyager or Maverick), and the cell product assessed for CD34⁺cell recovery, colony formation and viability.

TABLE 18 CD34+ cell recovery and sterility after sequential infusionsthrough the catheters. Catheter used Condition Parameter SprinterVoyager Maverick Pre-infusion CD34+ cell yield 9.72 × 10⁶ 2.11 × 10⁷5.73 × 10⁶ After 1^(st) CD34+ cell recovery 111%  103% 99% catheterAfter 2^(nd) CD34+ cell recovery 94% 104% 97% catheter After 3^(rd)CD34+ cell recovery 99%  99% 106%  catheter Sterility (aerobic andNegative Negative Negative anaerobic microbes)

As shown in Table 18, viable, colony forming cells were recovered in allexperiments for all three catheters tested (cell recovery 99%, 99% and106%).

As shown in Table 19, the average viability of the CD34+ cells afterpassing through the third catheter was 94.000% (based on a range of93.55%-94.40%) versus 96.01% (based on range of 94.18%-97.93%) of thepre-infusion cell product.

TABLE 19 CD34+ cell viability after sequential infusions through thecatheters. CD34+ cell viability Condition Sprinter Voyager MaverickAverage Pre-infusion 94.18% 95.91% 97.93% 96.01% After 1st catheter94.73% 96.31% 95.45% 95.50% After 2^(nd) Catheter 95.34% 95.72% 95.01%95.36% After 3rd catheter 93.55% 94.40% 94.04% 94.00%

As shown in Table 20, colony forming unit (CFU) growth derived from theCD34+ cells after passing through the third catheter was 95.27% (basedon a range of 43.47%-163.64%) of the infusion product (i.e., the infusedchemotactic hematopoietic stem cell product comprising CD34+ cells).

TABLE 20 CFU growth of CD34+ cells after sequential infusions throughthe catheters. CFU per 100 CD34+ cells cultured Condition SprinterVoyager Maverick Pre-infusion 30.5 11.5 11.0 After 1st catheter 22.014.0 22.0 After 2nd catheter 20.5 4.0 19.0 After 3rd catheter 24.0 5.018.0 Recovery from the pre- 78.69% 43.47% 163.64% infused product afterthe 3rd catheter Average recovery 95.27%

To determine the effect of catheter perfusion on CD34+ cell mobility andability to grow in culture, a series of experiments were performed whereMMH cells obtained from healthy donors were stored at 4° C. for 12 or 24hours before initiation of Isolex processing. Isolated CD34+ cellproduct that had been stored for about 12 hours pre-Isolex processingthen were stored at 4° C. until about 36 hours had elapsed from the endof processing, for a total of about 48 hours post MMH. At that time theywere assessed for SDF-1/CXCR4 mobility and CFU growth pre and postperfusion through a 0.36 mm inner diameter (i.d.) cardiac ballooncatheter. Similarly, cells that were stored pre-Isolex processing for 24hours then were stored at 4° C. until 48 hours had elapsed from the endof Isolex processing, for a total of 72 hours, and then assessed.

TABLE 21 12 inbound/48 outbound and 48 hour inbound/72 hour outboundfrom MMH: SDF-1/CXCR4 mobility (% population of migrated CD34+ cells)and CFU (per 100 viable CD34+ plated) pre catheter perfusion (“PRE”) andpost catheter perfusion (“POST”) Time (h) after MMH SDF-1/CXCR4 mobility(%) // # of Inbound/ CFU per 100 viable CD34+ cells plated outbound A BC D E 12/48 2.7 // 14  8.8 // 15 15.8 // 16 — — PRE 12/48 3.4 // 15 18.9// 13 17.6 // 8  — — POST 24/72 — — — 34 // 37 18.9 // 27.5 PRE 24/72 34// 43 23.5 // 24   POST

The results in Table 21 demonstrate that neither CD34+ CXCR-4-mediatedcell mobility nor the cell's ability to grow in culture at any of thetime points tested was affected adversely by perfusion through acatheter having an internal diameter of at least 0.36 mm.

The Stabilizing Effect of Serum

The following data confirm the importance of the stabilizing effect ofserum to the migratory capability of the selected CD34+ cells.

As shown in Table 22, no CXCR-4 migratory activity was observed for allsamples tested including the pre-catheter infusion samples when thecomposition comprising a chemotactic hematopoietic stem cell product wasformulated without serum.

TABLE 22 Chemotaxis of CD34+ cells after sequential infusions throughthe catheters in the absence of serum. Migration (%) Condition SprinterVoyager Maverick Pre-infusion 0.0 0.0 0.1 After 1st catheter 0.0 0.0 0.0After 2nd catheter 0.0 0.0 0.1 After 3rd catheter 0.0 0.0 0.0

FIGS. 2 and 3 further illustrate that Isolex selected CD34+ cells retaintheir migratory capacity longer when formulated in the presence of humanserum. Following Isolex processing, the bone marrow derivedhematopoietic stem cell product comprising selected CD34+ cells wasformulated either in (1) phosphate buffered saline (Dulbecco's phosphatebuffered saline, Ca++, Mg++Free (Baxter Cat. No. EDR9865) (“PBS”)containing 1% human serum albumin, 25 U/ml of heparin sodium and variousconcentrations (about 0%, about 10%, about 20%, or about 70%) ofautologous serum; or (2) normal saline (0.9%) containing 1% human serumalbumin, 25 U/ml of heparin sodium and (about 0% or about 10%)autologous serum. SDF-1/CXCR-4 mediated CD34+ cell migratory capacitywas evaluated at different times during final product storage (at 2°C.-8° C.) and after passing the cells through the catheter at the samerate and duration as anticipated by the clinical protocol. None of theseformulations affected CD34+ cell viability or the recovery of CD34+cells after they had been passed through the catheter.

Regardless of whether the chemotactic hematopoietic cell productscomprising selected CD34+ cells was (i) formulated either in PBS-serumor in saline-serum and (ii) either passed through the catheterimmediately or passed through the catheter after a prolonged stabilitytesting storage interval at about 4° C. to about 8° C., they maintainedan average of 96.6% viability (range 92.5%-98.6%) and an averageCXCR-4-mediated migratory capacity of 11.4% (range 2.4%-30.6%),representing a total time from harvest to mobility analysis of up to 48hours.

As shown in FIG. 2 panel (a), cells formulated in PBS alone at about 25hours retained about 10% of their CXCR-4 migratory capacity, whichdropped off to near 0 at about 48 hours. As shown in panel (b), cellsformulated in normal saline alone retained little, if any, of theirmigratory capacity. As shown in panels (c) and (d), cells formulatedwith PBS containing at least about 10% serum retained about 10-15% oftheir migratory capacity for up to about 55 hours (c), while cellsformulated with saline and at least about 10% serum retained about 20%of their migratory capacity for up to about 50 hours. As shown in panels(e) and (f), cells retained a higher migratory capacity for a longerduration in PBS supplemented with even higher concentrations of serum.

As shown in FIG. 3, the product of the present invention comprisingselected CD34+ cells when formulated in 10% serum, retained 14.25%, <1%,6%, and 5.8% of its CD34+CXCR4-mediated migratory capacity about 24,about 32, about 48 and about 56 hours after harvest, respectively. FIG.3 further shows that the product of the present invention comprisingselected CD34+ cells when formulated in 20% serum retained 18.25%,10.25%, 17% and 11% of its CD34+-CXCR4-mediated migratory capacity about24, about 32, about 48 and about 56 hours after harvest, respectively.The term “stabilizing amount” as used herein therefore refers to theamount of serum that, when included in the formulation of the product ofthe present invention comprising selected CD34+ cells, enables thesecells to retain their CXCR-4 mediated chemotactic activity andhematopoietic colony forming ability.

Study 5: Final Product. Sterility Testing

Due to the limited yield of CD34+ cells obtained from a 300-ml MMH,final cell product sterility will be assessed using the supernatantremoved from the final product formulation in order to preserve cellproduct for infusion. Supernatant samples are loaded into the syringesin a manner identical to that used to load the cell product into thesyringes used for infusion (see supra). To demonstrate that such asample will be representative of the final cell product formulation, weinoculated selected CD34+ cells in infusion solution prior tocentrifugation of the final product with C. sporogenes (13 CFU/ml), P.aeruginosa (2 CFU/ml), S. aureus (18 CFU/ml), A. niger (17 CFU/ml), C.albicans (3 CFU/ml) and B. subtilis (17 CFU/ml) (See table 22). Aftercentrifugation, the sterility of both cell pellet and non-cellsupernatant fractions was assessed using USP aerobic and anaerobictesting.

TABLE 23 Bacteria and fungi used for the sterility study. ExpectedCFU/ml of Total # of Total inoculated Microbe microbes/ml CFU/ml sample(21 ml) C. sporogenes 400 279 13 P. aeruginosa 400 36 2 S. aureus 400371 18 A. niger 400 356 17 C. albicans 400 62 3 B. subtilis 400 349 17Each source microorganism vial prepared by Microbiological Environmentscontained 400 microbes per ml, but the numbers of CFU derived from eachspecies are varied.

As shown in Table 24, both the cell pellet fraction and suspensionfractions from all tested samples showed outgrowth of the inoculatedmicroorganisms, while un-inoculated controls showed no growth. Further,no apparent differential growth rate was observed between testing ofcell pellet fractions and the suspension fractions for allmicroorganisms tested. Samples taken before each step of the processingprocedure and following the final perfusion through the catheters alltested negative for microbial contamination.

TABLE 24 14-day sterility testing of nucleated cell (NC) samplesinoculated with specific species of microorganism (400 microbes in 21-mlNC sample). Sample with microbe Medium Sample Inoculated type fractionTest 1 Test 2 Test 3 C. sporogenes FTM^(a) Cell pellet Positive PositivePositive Suspension Positive Positive Positive S. aureus FTM Cell pelletPositive Positive Positive Suspension Positive Positive Positive P.aeruginosa FTM Cell pellet Positive Positive Positive SuspensionPositive Positive Positive A. niger TSB^(b) Cell pellet PositivePositive Positive Suspension Positive Positive Positive C. albicans TSBCell pellet Positive Positive Positive Suspension Positive PositivePositive B. subtilis TSB Cell pellet Positive Positive PositiveSuspension Positive Positive Positive Positive control: C. sporogenesFTM Cell Positive Positive control: S. aureus FTM suspension PositivePositive control: P. aeruginosa FTM Positive Positive control: A. nigerTSB Positive Positive control: C. albicans TSB Positive Positivecontrol: B. subtilis TSB Positive Negative control: No microbes FTM CellNegative Negative control: No microbes TSB suspension Negative ^(a)Fluidthioglycollate medium ^(b)Tryptic soy broth

Preclinical Study Summary

Collectively, these preclinical data indicate that the manufacturing andtesting procedures described are capable of generating adequate numbersof viable cells with adequate stability to withstand shipment andperfusion through the catheter in a manner that should pose noadditional safety concerns to the subject other than those associatedwith the routine use of fluid infusion through the balloon catheter.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A pharmaceutical composition for repairing a vascular injury in a subject, wherein the vascular injury is an acute myocardial infarction resulting from a natural disease process and the subject is a revascularized subject, the pharmaceutical composition comprising: (a) a therapeutically effective amount of a sterile isolated chemotactic hematopoietic stem cell product, the chemotactic hematopoietic stem cell product comprising an enriched population of isolated CD34+ cells acquired from the subject and containing a subpopulation of potent cells having CXCR-4-mediated chemotactic activity; and (b) a stabilizing amount of serum, wherein the stabilizing amount of serum is greater than 20% (v/v), wherein the composition is administered to the subject parenterally through a catheter; and wherein the subpopulation of potent cells having CXCR-4-mediated chemotactic activity when passed through the catheter remains potent, is at least 70% pure, at least about 70% viable, is able to form hematopoietic colonies in vitro for at least about 24 hours following acquisition from the subject of a preparation comprising the enriched population of isolated CD34+ cells.
 2. The pharmaceutical composition according to claim 1, wherein the preparation comprising the enriched population of isolated CD34+ cells is purified from cellular components of a bone marrow aspirate acquired from the subject.
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. (canceled)
 7. The composition according to claim 1, wherein the therapeutically effective amount of the chemotactic hematopoietic stem cell product comprises a minimum number of isolated CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×10⁶ potent CD34+ cells having CXCR-4 mediated chemotactic activity.
 8. (canceled)
 9. The composition according to claim 1, wherein the enriched population of CD34+ cells is capable of forming hematopoietic colonies in vitro for at least 48 hours following acquisition and wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 48 hours following acquisition from the subject of the preparation comprising the enriched population of isolated CD34+ cells having CXCR-4-mediated chemotactic activity.
 10. The composition according to claim 1, wherein the enriched population of CD34+ cells is capable of forming hematopoietic colonies in vitro for at least 72 hours following acquisition and wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 72 hours following acquisition from the subject of the preparation comprising the enriched population of isolated CD34+ cells having CXCR-4-mediated chemotactic activity.
 11. (canceled)
 12. (canceled)
 13. The composition of claim 1, wherein at least 2% of the chemotactic activity of the subpopulation of potent cells is retained by the subpopulation of potent cells for at least 24 hours following acquisition from the subject of the preparation comprising the enriched population of isolated CD34+ cells having CXCR-4-mediated chemotactic activity.
 14. (canceled)
 15. (canceled)
 16. (canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. The composition according to claim 1, wherein the composition is administered through the catheter intravascularly to an infarct-related artery.
 21. The composition according to claim 1, wherein the catheter is a flow control catheter.
 22. The composition according to claim 1, wherein the catheter is a balloon catheter.
 23. The composition according to claim 1, wherein the catheter has an internal diameter of at least about 0.36 mm.
 24. (canceled)
 25. The composition according to claim 1 wherein the composition is administered through the catheter into myocardium.
 26. (canceled)
 27. The composition according to claim 1, wherein the pharmaceutical composition further includes at least one compatible active agent.
 28. (canceled)
 29. The composition according to claim 1, wherein sterility of the chemotactic hematopoietic cell product is confirmed by a method comprising the steps: (a) centrifuging the cell product to form (i) a separated cell product comprising a pellet comprising the enriched population of isolated CD34+ cells and (ii) a supernatant; (b) removing the supernatant of the separated cell product without disturbing the cell pellet of the separated cell product. (c) analyzing the sterility of the supernatant of the separated cell product, thereby determining the sterility of the cell pellet of the separated cell product without depleting the chemotactic hematopoietic cell product. 30-106. (canceled)
 107. (canceled)
 108. (canceled)
 109. The pharmaceutical composition according to claim 1, wherein the preparation comprising the enriched population of isolated CD34+ cells is purified from peripheral blood acquired from the subject. 